Oncolytic virus therapy for resistant tumors

ABSTRACT

Disclosed herein is a recombinant oncolytic virus comprising a nucleic acid sequence encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). One such oncolytic virus is oHSV. One form of TRAIL contained within the oncolytic virus is a secreted form of TRAIL. Examples of various forms of oHSV and secreted TRAIL are disclosed therein. Also disclosed are host cells and therapeutic formulations comprising the recombinant oncolytic virus. Also disclosed are methods of treating cancer in a subject by administering a therapeutic formulation comprising the recombinant oncolytic virus to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C § 120 ofco-pending U.S. application Ser. No. 14/416,754, filed on Jan. 23, 2015,which is a 35 U.S.C. § 371 National Phase Entry Application ofInternational Application No. PCT/US2013/031949 filed Mar. 15, 2013,which designates the U.S., and which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/675,013, filed Jul. 24,2012, the contents of each of which are incorporated herein by referencein their entirety.

GOVERNMENTAL SUPPORT

This invention was made with Government support under NS03677,CA138922-01, and NS076873 awarded by the National Institutes of Health.The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 3, 2017, isnamed 030258-073052-US_SL.txt and is 5,372 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the field of cancer therapeutics.

BACKGROUND OF THE INVENTION

Glioblastoma multiforme (GBM) is a high-grade glioma and the most commonprimary malignant brain tumor.¹ GBMs are diffuse and infiltrating withno clear border between normal brain and tumor. Current treatmentregimens that include temozolomide have significantly improved themedian, 2- and 5-year survival compared to radiotherapy alone inpatients with newly diagnosed GBM.^(2,3) Nevertheless, GBM patients havea poor prognosis with a median survival of 14.6 months.² The inherent oracquired resistance of tumor cells to antitumor agents and the highlyinvasive nature of tumor cells are the major impediments to thecurrently employed anti-GBM therapies and pose an urgent need for noveltherapeutics with substantial efficacy. Oncolytic herpes simplex virus(oHSV) and TRAIL (tumor necrosis factor-related apoptosis-inducingligand) have recently shown promise in both preclinical and clinicaltrials.^(4,5,6,7,8,9,10,11,12,13) Oncolytic viruses are geneticallymodified viruses that, upon infection, selectively replicate in and killneoplastic cells while sparing normal cells.^(4,8,14) Among them, oHSVtype 1-derived virus is one of the most extensively studied andconsidered a promising agent for treating brain tumors as well as othertypes of cancer.^(4,15) Recombinant oHSV vectors such as G207 and G47Δhave been previously investigated in both preclinical and clinicalstudies.^(9,16,17,18) Unlike replication-incompetent vectors,replication-competent or conditional vectors can amplify to producevirus progeny that then infects surrounding tumor cells resulting inmultiple waves of infection in situ, virus spread and extensive celldeath. In a direct comparison between oncolytic adenovirus and oHSV inGBM cell lines, oHSV was shown to be more efficacious.¹⁹ Mutations ofspecific HSV genes, namely γ34.5 and UL39, have been shown to conferselectivity to cancer cells, which has enabled translational studies tohumans.^(4,15) Although phase 1 and 1b clinical trials for oHSV provedits safety, the efficacy for human GBMs seems marginal as only a subsetof patients showed decrease in tumor volume⁹ which could in part be dueto the insensitivity of a subset of GBM cells to HSV mediated oncolysis.

TRAIL has emerged as a promising antitumor agent due to itstumor-specific induction of apoptosis in a death receptor-dependentmanner.²⁰ Both recombinant human TRAIL ligand and TRAIL receptor agonistmonoclonal antibodies are currently being evaluated in clinicaltrials,²¹ however, short half-life and off-target toxicity ofsystemically delivered TRAIL pose challenges in the clinic.²² It haspreviously been established that a secreted form of TRAIL (S-TRAIL)exerts more potent apoptotic effects compared to TRAIL itself and whendelivered by viruses or different stem cell types has significantantitumor effects as compared to systemically administrated TRAIL indifferent mouse models of GBMs.^(5,7,10,11,12,23) However, malignantGBMs show heterogeneity in their response to TRAIL; with ˜50% showingsensitivity to TRAIL-mediated apoptosis and others showing varyingresistance to TRAIL-mediated apoptosis.

SUMMARY OF THE INVENTION

Aspects of the invention relate to a recombinant oncolytic viruscomprising a nucleic acid sequence encoding tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) or a biologicallyactive fragment thereof, in expressible form. In one embodiment, theoncolytic virus is an oncolytic herpes simplex virus (oHSV). In oneembodiment, the oHSV is G207, G47Δ, HSV-R3616, 1716, R3616, or R4009. Inone embodiment, the TRAIL is a secreted form of TRAIL (S-TRAIL). In oneembodiment, the TRAIL is a TRAIL fusion protein. In one embodiment, theTRAIL is regulated by the HSV immediate early 4/5 promoter. In oneembodiment, the oncolytic virus contains an additional exogenous nucleicacid in expressible form. In one embodiment, the virus contains noadditional exogenous nucleic acids.

Another aspect of the invention relates to a nucleic acid comprising thegenome of a recombinant oncolytic virus described herein. In oneembodiment, the nucleic acid is a bacterial artificial chromosome (BAC),a P1-derived artificial chromosome (PAC), a yeast artificial chromosome(YAC) or a human artificial chromosome (HAC). Another aspect of theinvention relates to a host cell comprising a recombinant oncolyticvirus described herein or the nucleic acid comprising the genome of therecombinant oncolytic virus.

Another aspect of the invention relates to a pharmaceutical compositioncomprising the recombinant oncolytic virus described herein. Anotheraspect of the invention relates to a kit comprising the pharmaceuticalcomposition described herein, and instructions for use.

Another aspect of the invention relates to a method of inhibiting tumorprogression in a subject comprising contacting the tumor with aneffective amount of a recombinant oncolytic virus described herein. Inone embodiment, the tumor is a brain tumor. In one embodiment, the braintumor is a glioma. In one embodiment the tumor is malignant. In oneembodiment the tumor is selected from the group consisting of adenoma,angio-sarcoma, astrocytoma, epithelial carcinoma, germinoma,glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma,hematoma, hepato-blastoma, leukemia, lymphoma, medulloblastoma,melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma,sarcoma, and teratoma. The tumor can be chosen from acral lentiginousmelanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma,adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors,bartholin gland carcinoma, basal cell carcinoma, bronchial glandcarcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous,cholangio-carcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodularhyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma,hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma,hepatic adenomatosis, epatocellular carcinoma, insulinoma,intaepithelial neoplasia, interepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,lentigo maligna melanomas, malignant melanoma, malignant mesothelialtumors, medulloblastoma, medulloepithelioma, melanoma, meningeal,mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cellcarcinoma, oligodendroglial, osteosarcoma, pancreatic, papillary serousadeno-carcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudo-sarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor. In one embodiment contacting is by a method of administration tothe subject is by a method selected from the group consisting ofintravenous administration, intraperitoneal administration,intramuscular administration, intracoronary administration,intraarterial administration, subcutaneous administration, transdermaldelivery, intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation, intracerebral, nasal, oral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue or tumor.

Another aspect of the invention relates to a method of treating cancerin a subject comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising therecombinant oncolytic virus described herein to thereby treat thecancer. In one embodiment, the cancer is selected from the groupconsisting of basal cell carcinoma, biliary tract cancer; bladdercancer; bone cancer; brain and CNS cancer; breast cancer; cancer of theperitoneum; cervical cancer; choriocarcinoma; colon and rectum cancer;connective tissue cancer; cancer of the digestive system; endometrialcancer; esophageal cancer; eye cancer; cancer of the head and neck;gastric cancer (including gastrointestinal cancer); glioblastoma (GBM);hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renalcancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung); lymphoma includingHodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma;oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovariancancer; pancreatic cancer; prostate cancer; retinoblastoma;rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer;stomach cancer; testicular cancer; thyroid cancer; uterine orendometrial cancer; cancer of the urinary system; vulval cancer; as wellas other carcinomas and sarcomas; as well as B-cell lymphoma (includinglow grade/follicular non-Hodgkin's lymphoma (NEIL); small lymphocytic(SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuseNHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; highgrade small non-cleaved cell NHL; bulky disease NHL; mantle celllymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia);chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. In one embodiment,the cancer is brain cancer. In one embodiment, the brain cancer isglioma or glioblastoma. In one embodiment, administration is by a methodselected from the group consisting of intravenous administration,intraperitoneal administration, intramuscular administration,intracoronary administration, intraarterial administration, subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation, intracerebral, nasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue or tumor.

Definitions

An “oncolytic virus” is any virus which typically is able to kill atumor cell (non-resistant) by infecting the tumor cell.

An oncolytic virus is “replication-selective” if it is more capable ofreplicating or is capable of replicating to a greater extent (e.g. burstsize) in a tumor cell of a subject than in a non-tumor cell of thesubject.

The term “in expressible form” when used in the context of a DNAmolecule means operably linked (e.g., located within functionaldistance) to sequences necessary for transcription of the DNA into RNAby the RNA polymerase transcription machinery found in eukaryotic cells(e.g., promoter sequences, and other 5′ regulatory sequences). Oneexample is a DNA molecule in the context of an expression vector.Expression can refer to transcription of DNA into RNA, and when proteincoding sequences are involved, expression may also encompass translationof the mRNA into protein. Viral expression vectors may comprise theviral genome in the context of a virion that is used to infect a cell.

The term “operably linked” is used herein to refer to a functionalrelationship of one nucleic acid sequence to another nucleic acidsequence. Nucleic acid sequences are “operably linked” when placed intoa functional relationship with one another. For example, a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. The DNA sequences being linked may be contiguous, orseparated by intervening sequences, and when necessary in the samereading phase and/or appropriate orientation. Linking is accomplished,for example, by ligation at convenient restriction sites. If such sitesdo not exist, the synthetic oligonucleotide adaptors or linkers are usedin accordance with conventional practice.

The term “heterologous” is used herein to describe the relationship ofone nucleic acid or amino acid sequence to one or more different nucleicacid or amino acid sequences, respectively. The term heterologous, inreference to two or more such sequences, indicates that the differentsequences are found in nature within separate, different and distinctlarger nucleic acids or polypeptides. The joining of heterologoussequences creates a non-naturally occurring juxtaposition of sequences.Such joining is the product of engineering performed in the laboratory.When such amino acid sequences are joined, the resulting protein isreferred to herein as a fusion protein. The products of such joining arereferred to as “recombinant”.

The term “isolated” when used in reference to a nucleic acid sequencerefers to the fact that the nucleic acid sequence is removed from thecontext of other nucleic acid sequences in which it is present in nature(e.g., in the context of a chromosome). The nucleic acids of theinvention are typically present in isolated form.

The term “purified” when used in reference to a polypeptide or virusrefers to the fact that it is removed from the majority of othercellular components from which it was generated or in which it istypically present in nature. The polypeptides and viruses describedherein may be in a state where they are purified or semi-purified.

As the term is used herein, “transfection” refers to the introduction ofnucleic acid into a cell (e.g, for the purpose of propagation and/orexpression of the nucleic acid by the cell). Examples of methods oftransfection are electroporation, calcium phosphate, lipofection, andviral infection utilizing a viral vector. Often nucleic acid isintroduced into a cell in expressible form. That means that the nucleicacid is in the appropriate context of regulatory sequences such that thecellular machinery will recognize it and process it (e.g., transcribeRNA from DNA, translate protein from RNA). In one embodiment, a nucleicacid is in expressible form when it is inserted into an expressionvector in the proper orientation to confer expression.

An “effective amount” as the term is used herein, is used to refer to anamount that is sufficient to produce at least a reproducibly detectableamount of the desired results. An effective amount will vary with thespecific conditions and circumstances. Such an amount can be determinedby the skilled practitioner for a given situation.

The term “therapeutically effective amount” refers to an amount that issufficient to produce a therapeutically significant reduction in one ormore symptoms of the condition when administered to a typical subjectwho has the condition. A therapeutically significant reduction in asymptom is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 100%, or more ascompared to a control or non-treated subject.

The term “treat” or “treatment” refers to therapeutic treatment whereinthe object is to eliminate or lessen symptoms. Beneficial or desiredclinical results include, but are not limited to, elimination ofsymptoms, alleviation of symptoms, diminishment of extent of condition,stabilized (i.e., not worsening) state of condition, delay or slowing ofprogression of the condition.

The terms “patient”, “subject” and “individual” are used interchangeablyherein, and refer to an animal, particularly a human, to whom treatmentincluding prophylaxic treatment is provided. This includes human andnon-human animals. The term “non-human animals” and “non-human mammals”are used interchangeably herein includes all vertebrates, e.g., mammals,such as non-human primates, (particularly higher primates), sheep, dog,rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows,and non-mammals such as chickens, amphibians, reptiles etc. In oneembodiment, the subject is human. In another embodiment, the subject isan experimental animal or animal substitute as a disease model. Patientor subject includes any subset of the foregoing, e.g., all of the above,but excluding one or more groups or species such as humans, primates orrodents. A subject can be male or female. A subject can be a fullydeveloped subject (e.g., an adult) or a subject undergoing thedevelopmental process (e.g., a child, infant or fetus).

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with unwanted neuronal activity. In addition, themethods and compositions described herein can be used to treatdomesticated animals and/or pets.

The term “mammal” refers to any animal classified as a mammal, includinghumans, non-human primates, domestic and farm animals, and zoo, sports,or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder is atumor. In one embodiment, the cell proliferative disorder is cancer.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein. In one embodiment, tumors arebenign. Examples of benign tumors include, without limitation,schwannomas, lipoma, chondroma, adenomas (e.g, hepatic adenoma), andbenign brain tumors (e.g., glioma, astrocytoma, meningioma).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

The term “inhibiting tumor cell growth or proliferation” meansdecreasing A tumor cell's growth or proliferation by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducingcell death in a cell or cells within a cell mass.

The term “tumor progression” refers to all stages of a tumor, includingtumorigenesis, tumor growth and proliferation, invasion, and metastasis.

The term “inhibiting tumor progression” means inhibiting thedevelopment, growth, proliferation, or spreading of a tumor, includingwithout limitation the following effects: inhibition of growth of cellsin a tumor, (2) inhibition, to some extent, of tumor growth, includingslowing down or complete growth arrest; (3) reduction in the number oftumor cells; (4) reduction in tumor size; (5) inhibition (i.e.,reduction, slowing down or complete stopping) of tumor cell infiltrationinto adjacent peripheral organs and/or tissues; (6) inhibition (i.e.reduction, slowing down or complete stopping) of metastasis; (7)increase in the length of survival of a patient or patient populationfollowing treatment for a tumor; and/or (8) decreased mortality of apatient or patient population at a given timepoint following treatmentfor a tumor.

A tumor “responds” to a particular agent if tumor progression isinhibited as defined above.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, bacterial artificial chromosomes(BAC), P1-derived artificial chromosome (PAC), yeast artificialchromosome (YAC), human artificial chromosome (HAC), DNA or RNAexpression vectors associated with cationic condensing agents, DNA orRNA expression vectors encapsulated in liposomes, and certain eukaryoticcells, such as producer cells. Expression may be achieved in anyappropriate host cell that has been transformed, transfected or infectedwith the expression vector. Suitable host cells include prokaryotes,yeast and higher eukaryotic cells. Preferably the host cells employedare E. coli, yeast or a mammalian cell, such as a primary cell or cellline such as COS or CHO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D contain graphical representations of experimental resultsthat indicate differential sensitivities of glioblastoma multiforme(GBM) cells to S-TRAIL-mediated apoptosis and oncolytic herpes simplexvirus (oHSV)-mediated oncolysis. FIG. 1A Screening of different GBMlines reveals differential sensitivities to S-TRAIL-mediated apoptosisand oHSV-mediated oncolysis. Established GBM lines (U87, LN229, U251,and Gli36) and primary glioma stem cell (GSC) lines (BT74, GBM4, GBM6,and GBM8F) were treated with different concentrations of purifiedS-TRAIL and assayed for viability at 48 hours post-treatment (top row),and for caspase-3/7 activity at 18 hours post-treatment (second row).Established GBM lines and primary GSC lines were infected with oHSV atmultiplicities of infection (MOIs) 0.2 and 1 and assayed for viabilityat 72 hours postinfection (third row), and virus titration on Vero cellsusing the supernatant of oHSV-infected GBM cell lines (MOI1, bottomrow). FIGS. 1B, 1C. Pharmacodynamics of oHSV in vitro. LN229-RmC andGBM8F-RmC were infected with oHSV-Fluc at MOI=1. Viral replicationindicated by (FIG. 1B) firefly luciferase (Fluc) activity and tumor cellviability indicated by (FIG. 1C) Renilla luciferase (Rluc) activity weremonitored by dual Fluc and Rluc bioluminescence imaging, respectively atdifferent time points. FIG. 1D. Pharmacodynamics of oHSV in vivo. Mice-(n=3 per line) bearing intracranial LN229-RmC (left) or GBM8F-RmC(right) GBMs were injected with oHSV-Fluc intratumorally and viraldistribution was followed by Fluc bioluminescence imaging at differentindicated times. One representative image of mice and the average Flucbioluminescence intensities are shown. *P<0.05. Error bars indicate SD.

FIGS. 2A-2F contain graphical representations of experimental resultsthat indicate Combination of oncolytic herpes simplex virus (oHSV) andS-TRAIL leads to caspase-3/7-mediated apoptosis in resistantglioblastoma multiforme (GBM) cells. FIGS. 2A-Caspase-3/7 activity andcell viability of (FIGS. 2A, 2C) LN229 and (FIGS. 2B, 2D) GBM8F treatedwith purified S-TRAIL (100 ng/ml), oHSV (multiplicity of infection(MOI)=1) or both oHSV and S-TRAIL in the presence or absence ofpan-caspase inhibitor, Z-VAD-FMK (20 μmol/l). *P<0.05 in the comparisonof oHSV plus S-TRAIL treatment group with other groups. FIG. 2E, 2F.Immunoblot analysis using cleaved poly-ADP ribose polymerase (PARP)antibody on whole cell lysates prepared from LN229 and GBM8F cellstreated with purified S-TRAIL (100 ng/ml), oHSV (MOI=1) or both oHSV andS-TRAIL in the presence or absence of pan-caspase inhibitor, Z-VAD-FMK(20 μmol/l). Cleaved PARP expression was normalized to α-tubulinexpression. *P<0.05 in the comparison of oHSV plus tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) to TRAIL. Error barsindicate SD.

FIGS. 3A-3C contain graphical representations of experimental resultsthat indicate Oncolytic herpes simplex virus (oHSV)-tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) mediates potentcytotoxicity in resistant glioblastoma multiforme (GBM) cells byaltering both cell proliferation and death pathways in vitro. FIGS. 3A,3B. Cell viability of GBM cells assessed by Renilla luciferase (Rluc)bioluminescence at different time points. (FIG. 3A) LN229-RmC and (FIG.3B) GBM8F-RmC were infected with oHSV or oHSV-TRAIL at multiplicity ofinfection (MOI)=1. *P<0.05 in the comparison of oHSV-TRAIL to controland to oHSV. Error bars indicate SD. FIG. 3C. Immunoblot analysis usingantibodies against caspase-8, -9, cleaved poly-ADP ribose polymerase(PARP), Bcl2, phospho-ERK, ERK, phospho-c-Jun N-terminal kinase (JNK),JNK, phospho-p38, and p38 on whole cell lysates prepared from LN229 andGBM8F cells untreated, or treated with oHSV, oHSV-TRAIL (MOI=1) orS-TRAIL for 18 hours. α-Tubulin was used as a loading control.

FIGS. 4A-4D contain graphs and photographs of experimental results thatindicate Oncolytic herpes simplex virus (oHSV)-tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis inresistant glioblastoma multiformes (GBMs) depends on c-Jun N-terminalkinase (JNK) activation and ERK inhibition. FIG. 4A. Immunoblot analysisusing antibodies against JNK, phosho-JNK, and cleaved poly-ADP ribosepolymerase (PARP), on whole cell lysate prepared from LN229 cellstreated with oHSV, oHSV-TRAIL (multiplicity of infection (MOI)=1) orcontrol in the absence (−) and presence (+) of JNK inhibitor (SP600125,20 μmol/l) for 18 hours. FIG. 4B. Caspase 3/7 activity of LN229 cellstreated with oHSV, oHSV-TRAIL (MOI=1), or control for 20 hours in theabsence (−) and presence (+) of JNK inhibitor. *P<0.05 in the comparisonof oHSV-TRAIL treated cells in the absence and presence of JNK. FIG. 4C.Immunoblot analysis using antibodies against ERK, phosho ERK, andcleaved PARP on whole cell lysate prepared from LN229 cells treated withS-TRAIL (100 ng/ml) in the absence (−) and presence (+) of ERK inhibitor(U0126, 20 μmol/l) for 18 hours. α-Tubulin was used as a loadingcontrol. FIG. 4D. Cell viability assay showing the % viable LN229 cellsafter treatment with different combinations of U0126 (20 μmol/l) andTRAIL (100 ng/ml) for 48 hours. *P<0.05 JNK inhibitor group in thecomparison with control (in FIG. 4B) and ERK inhibitor and S-TRAILtreatment group in the comparison with other treatment groups (in FIG.4D). Error bars indicate SD.

FIGS. 5A-5D contain graphs and photographs of experimental results thatindicate Oncolytic herpes simplex virus (oHSV)-tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) prolongs survival ofmice-bearing both TRAIL and oHSV resistant glioblastoma multiforme(GBM). FIG. 5A. Timeline and survival curves of LN229-FmC GBM-bearingmice treated with oHSV, oHSV-TRAIL, or control (phosphate-bufferedsaline (PBS)). P=0.038 in oHSV and oHSV-TRAIL comparison, log-rank test.FIG. 5B. X-gal staining revealing virus-infected areas. oHSV-injected(left) and oHSV-TRAIL-injected (right) tumor sections. Originalmagnification ×20. FIG. 5C. Immunofluorescence of cleaved caspase-3staining on brain sections from LN229-FmC GBM-bearing mice injected withoHSV, oHSV-TRAIL, or PBS (control). Original magnification ×20. FIG. 5D.Plot showing the percentage of cleaved caspase-3 positive LN229-FmC GBMcells on brain sections. *P<0.05 in the comparison of oHSV-TRAIL tocontrol and to oHSV. n=3 in each group. Error bars indicate SD.

FIGS. 6A-6B contain graphs and photographs of experimental results thatindicate Oncolytic herpes simplex virus (oHSV)-tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) inhibits glioblastomamultiforme (GBM) invasion in vitro and in vivo in TRAIL resistant GBM.FIG. 6A. In vitro invasion assay. Photomicrographs and graph showing thechange in cell invasion after treatment with oHSV or oHSV-TRAIL in GBM8Fglioma line. Multiplicity of infection (MOI)=1. *P<0.05 in thecomparison of oHSV-TRAIL to control and to oHSV. FIG. 6B. In vivoinvasion assay. Mice-bearing intracranial GBM8-FmC gliomas were injectedwith oHSV, oHSV-TRAIL, or phosphate-buffered saline (PBS) (control) andmice were sacrificed on day 14 and invasion of the GBM cells on brainsections was evaluated. Photomicrograph of hematoxylin and eosin (H&E)staining of GBM8F tumor cell invasion towards adjacent normal braintissue and illustration of brain revealing GBM8F implantation-site andpictured-site. n=3 per group. *P<0.05 in the comparison of oHSV-TRAIL tocontrol and to oHSV. Error bars indicate SD.

FIG. 7 is a Schematic presentation showing the mechanism underlying theefficacy of oncolytic herpes simplex virus (oHSV)-tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) on resistantglioblastoma multiforme (GBM) cells.

FIG. 8 is a schematic of oHSV bearing Fluc or S-TRAIL transgene. TheHSV-1 genome consists of long and short unique regions (UL and Us) eachbounded by terminal (T) and internal (I) repeat regions (R_(L) andR_(S)). G47Δ-TRAIL (oHSV-TRAIL), and G47Δ-Fluc (oHSV-Fluc) weregenerated from G47ΔBAC, which is derived from the third-generationoncolytic HSV-1 (G47Δ) with triple mutations (γ34.5, ICP6, α47). Thetransgene Fluc or S-TRAIL as well as lacZ are located in the ICP6 locusin respective recombinants.

FIGS. 9A-9B contain graphs of experimental results that indicatePharmacodynamics of oHSV in vitro at different MOIs. FIG. 9A. Viralreplication monitored by Fluc bioluminescence imaging inLN229-Rluc-mCherry and GBM8F-Rluc-mCherry cells infected with oHSV-Flucat various MOIs (0-2). Imaging was performed 48 hours post infection.FIG. 9B. Tumor cell viability monitored by Rluc bioluminescence imagingin LN229-Rluc-mCherry and GBM8F-Rluc-mCherry cells infected withoHSV-Fluc at various MOIs (0-2). Imaging was performed 48 hours postinfection. Error bars indicate standard deviation. *, P<0.05.

FIGS. 10A-10E contain graphs of experimental results that indicateoHSV-TRAIL virus yield is similar to oHSV yield and mediates potentcytotoxicity in resistant GBM cells. FIG. 10A. oHSV, oHSV-Fluc andoHSV-TRAIL infected GBM cells at MOI=0.8 were harvested 24 hours afterinfection and the virus yield was quantified using plaque assay on Verocells. FIG. 10B. The concentrations of S-TRAIL determined by ELISA inconditioned media of LN229 GBM cells infected with oHSV-TRAIL atdifferent MOIs. FIGS. 10C-10D. Cell viability of LN229-Rluc-mCherrycells (FIG. 10C) and GBM8F-Rluc-mCherry cells (FIG. 10D) infected withoHSV or oHSV-TRAIL assessed by Rluc bioluminescence imaging 48 hourspost infection. Error bars indicate standard deviation. *, p<0.05. FIG.10E. FACS analysis showing the percentage of apoptotic cells (Annexin Vpositive, PI negative) following infection with oHSV or oHSV-TRAIL for24 hours.

FIG. 11 is a graph of experimental results that indicate oHSV-TRAILmediates potent cytotoxicity in sensitive GBM cells. Gli36 cellsinfected with oHSV or oHSV-TRAIL were assayed for viability at 48 hourspost infection. Error bars indicate standard deviation. *, p<0.05

FIG. 12 contains photographs of experimental results that indicate oHSVTRAIL mediated potent cytotoxicity is independent of DR4/DR5 expression:Immunoblot analysis using antibodies against DR4 and DR5 on whole celllysates prepared from LN229 and GBM8F cells untreated, or treated withoHSV, oHSV-TRAIL (MOI=1) or S-TRAIL for 18 hours. α-tubulin was used asa loading control.

FIG. 13 contains graphs and photographs of experimental results thatindicate oHSV-TRAIL inhibits growth of both TRAIL and oHSV resistantLN229-Fluc-mCherry GBMs in vivo: Mice bearing LN229-Fluc-mCherry GBMstreated with oHSV, oHSV-STRAIL or PBS (control) were followed forchanges in tumor volumes by Fluc bioluminescence intensities. Onerepresentative image of mice and the average tumor volume of each groupon 7 days after treatment are shown. The average tumor volumes werenormalized to the control (PBS) group. n=5 in each group. * p<0.05 inthe comparison of oHSV-TRAIL to control and to oHSV. Error bars indicatestandard deviation.

FIG. 14 contains photographs of experimental results that indicate H&Estained mouse brain sections harboring intracerebral tumors. Left panel,GBM8F with arrows showing contralateral tumor extension. Right panel,LN229. Arrows show discrete tumor brain borders.

DETAILED DESCRIPTION OF THE INVENTION

Only a subset of cancer patients inoculated with oncolytic herpessimplex virus (oHSV) type-1 has shown objective response in phase 1 and2 clinical trials. This has raised speculations whether resistance oftumor cells to oHSV therapy may be a limiting factor. In the experimentsdisclosed herein, established and patient derived primary glioblastomamultiforme (GBM) stem cell lines (GSC) resistant to oHSV and also totumor necrosis factor-related apoptosis-inducing ligand (TRAIL) wereidentified that had recently shown promise in preclinical and initialclinical studies. A recombinant oHSV bearing a secretable TRAIL(oHSV-TRAIL) was created and used to test the hypothesis that oHSV-TRAILcould be used as a cancer therapeutic to target a broad spectrum ofresistant tumors in a mechanism-based manner. Using the identifiedresistant GBM lines, oHSV-TRAIL was shown to downregulate extracellularsignal-regulated protein kinase (ERK)-mitogen-activated protein kinase(MAPK) and upregulate c-Jun N-terminal kinase (JNK) and p38-MAPKsignaling, which primed resistant GBM cells to apoptosis via activationof caspase-8, -9, and -3. Further, it was shown that oHSV-TRAILinhibited tumor growth and invasiveness and increased survival of micebearing resistant intracerebral tumors without affecting the normaltissues. This study sheds new light on the mechanism by which oHSV andTRAIL function in concert to overcome therapeutic-resistance, andprovides an oncolytic virus based platform to target a broad spectrum ofdifferent cancer types. Aspects of the invention relate to the discoveryof this oncolytic virus based platform.

One aspect of the invention relates to a recombinant oncolytic virusthat comprises a heterologous nucleic acid sequence encoding TRAIL, or abiologically active fragment thereof, in expressible form. Anotheraspect of the invention relates to a nucleic acid comprising the genomeof the oncolytic virus. Such a nucleic acid may be in the form of anartificial chromasome, such as a bacterial artificial chromosome (BAC),a P1-derived artificial chromosome (PAC), a yeast artificial chromosome(YAC), or a human artificial chromosome (HAC). The invention alsoencompasses a host cell or population thereof, comprising the oncolyticvirus and/or the nucleic acid comprising the genome of the oncolyticvirus.

Oncolytic Viruses

Numerous oncolytic viruses are known in the art and are described, forexample, in Kirn et al. (1999, In: Gene Therapy of Cancer, AcademicPress, San Diego, Calif., pp. 235-248), any of which is envisioned foruse in the invention. By way of example, appropriate oncolytic virusesinclude type 1 herpes simplex viruses, type 2 herpes simplex viruses,vesicular stomatitis viruses, oncolytic adenovirus (U.S. Pat. No.8,216,819), Newcastle disease viruses, vaccinia viruses, and mutantstrains of these viruses. In one embodiment, the oncolytic virus isreplication-selective or replication-competent. In one embodiment, theoncolytic virus is replication-incompetent.

The oncolytic viruses useful in the present methods and compositionsare, in some embodiments, replication-selective. It is understood thatan oncolytic virus may be made replication-selective if replication ofthe virus is placed under the control of a regulator of gene expressionsuch as, for example, the enhancer/promoter region derived from the5′-flank of the albumin gene (e.g. see Miyatake et al., 1997, J. Virol.71:5124-5132). By way of example, the main transcriptional unit of anHSV may be placed under transcriptional control of the tumor growthfactor-beta (TGF-β) promoter by operably linking HSV genes to the TGF-βpromoter. It is known that certain tumor cells overexpress TGF-β,relative to non-tumor cells of the same type. Thus, an oncolytic viruswherein replication is subject to transcriptional control of the TGF-βpromoter is replication-selective, in that it is more capable ofreplicating in the certain tumor cells than in non-tumor cells of thesame type. Similar replication-selective oncolytic viruses may be madeusing any regulator of gene expression which is known to selectivelycause overexpression in an affected cell. The replication-selectiveoncolytic virus may, for example, be an HSV-1 mutant in which a geneencoding ICP34.5 is mutated or deleted.

An oncolytic virus in accordance with the present invention can furthercomprise other modifications in its genome. For example, it can compriseadditional DNA inserted into the UL44 gene. This insertion can producefunctional inactivation of the UL44 gene and the resulting lyticphenotype, or it may be inserted into an already inactivated gene, orsubstituted for a deleted gene.

The oncolytic virus may also have incorporated therein one or morepromoters that impart to the virus an enhanced level of tumor cellspecificity. In this way, the oncolytic virus may be targeted tospecific tumor types using tumor cell-specific promoters. The term“tumor cell-specific promoter” or “tumor cell-specific transcriptionalregulatory sequence” or “tumor-specific promoter” or “tumor-specifictranscriptional regulatory sequence” indicates a transcriptionalregulatory sequence, promoter and/or enhancer that is present at ahigher level in the target tumor cell than in a normal cell. Forexample, the oncolytic virus for use in the invention may be under thecontrol of an exogenously added regulator such as tetracycline (U.S.Pat. No. 8,2366,941).

In one embodiment, the oncolytic virus (e.g, oHSV) vector of theinvention is engineered to place at least one viral protein necessaryfor viral replication under the control of a tumor-specific promoter.Or, alternatively a gene (a viral gene or exogenous gene) that encodes acytotoxic agent can be put under the control of a tumor-specificpromoter. By cytotoxic agent as used here is meant any protein thatcauses cell death. For example, such would include ricin toxin,diphtheria toxin, or truncated versions thereof. Also, included would begenes that encode prodrugs, cytokines, or chemokines. Such viral vectorsmay utilize promoters from genes that are highly expressed in thetargeted tumor such as the epidermal growth factor receptor promoter(EGFr) or the basic fibroblast growth factor (bFGF) promoter, or othertumor associated promoters or enhancer elements.

Oncolytic Herpes Simplex Virus (oHSV)

One such oncolytic virus for use in the present invention is oncolyticherpes simplex virus (oHSV). The oHSV will comprise one or moreexogenous nucleic acids encoding for one or more of the polypeptidesdescribed herein. Methods of generating an oHSV comprising such anexogenous nucleic acid are known in the art. The specific position ofinsertion of the nucleic acid into the oHSV genome can be determined bythe skilled practitioner.

Oncolytic herpes simplex viruses (oHSV) are known in the art and aredescribed, for example, in Kim et al. (1999, In: Gene Therapy of Cancer,Academic Press, San Diego, Calif., pp. 235-248), and include type 1herpes simplex viruses and type 2 herpes simplex viruses. In oneembodiment, the oHSV used in the methods, compositions, and kits of theinvention is replication-selective or replication-competent such as oneof the examples described herein. In one embodiment, the oHSV isreplication-incompetent.

Herpes simplex 1 type viruses are among the preferred viruses, forexample the variant of HSV-1 viruses that do not express functionalICP34.5 and thus exhibit significantly less neurotoxicity than theirwild type counterparts. Such variants include without limitationoHSV-R3616, one of the HSV-1 viruses described in Coukos et al., GeneTher. Mol. Biol. 3:79-89 (1998), and Varghese and Rabkin, Cancer GeneTherapy 9:967-978 (2002). Other exemplary HSV-1 viruses include 1716,R3616, and R4009. Other replication selective HSV-1 virus strains thatcan be used include, e.g., R47Δ (wherein genes encoding proteins ICP34.5and ICP47 are deleted), G207 (genes encoding ICP34.5 and ribonucleotidereductase are deleted), NV1020 (genes encoding UL24, UL56 and theinternal repeat are deleted), NV1023 (genes encoding UL24, UL56, ICP47and the internal repeat are deleted), 3616-UB (genes encoding ICP34.5and uracil DNA glycosylase are deleted), G92Δ (in which the albuminpromoter drives transcription of the essential ICP4 gene), hrR3 (thegene encoding ribonucleotide reductase is deleted), and R7041 (Us3 geneis deleted) and HSV strains that do not express functional ICP34.5.

oHSV for use in the methods and compositions described herein is notlimited to one of the HSV-1 mutant strains described herein. Anyreplication-selective strain of a herpes simplex virus may be used. Inaddition to the HSV-1 mutant strains described herein, other HSV-1mutant strains that are replication selective have been described in theart. Furthermore, HSV-2, mutant strains such as, by way of example,HSV-2 strains 2701 (RL gene deleted), Delta RR (ICP10PK gene isdeleted), and FusOn-H2 (ICP10 PK gene deleted) can also be used in themethods and compositions described herein.

Non-laboratory strains of HSV can also be isolated and adapted for usein the invention (U.S. Pat. No. 7,063,835). Furthermore, HSV-2 mutantstrains such as, by way of example, HSV-2 strains HSV-2701, HSV-2616,and HSV-2604 may be used in the methods of the invention.

In a one embodiment, the oHSV is G47Δ. G47Δ is a third generation virus,which contains 1) a mutation of ICP6, which targets viral deletion totumor cells, 2) a deletion of γ34.5, which encodes ICP34.5 and blockseIF2a phosphorylation and is the major viral determinant ofneuropathogenicity, and 3) an additional deletion of the ICP47 gene andUS11 promoter, so that the late gene US11 is now expressed as animmediate-early gene and able to suppress the growth inhibitedproperties of γ34.5 mutants. Deletion of ICP47 also abrogates HSV-1inhibition of the transporter associated with antigen presentation andMEW class 1 downregulation (Todo et al., Proc. Natl. Acad. Sci. USA,98:6396-6401(2001)).

TRAIL

The oncolytic virus described herein comprises a nucleic acid sequencethat encodes TRAIL, or a biologically active fragment thereof,incorporated into the virus genome in expressible form. As such theoncolytic virus serves as a vector for delivery of TRAIL to the infectedcells. The invention envisions the use of various forms of TRAIL, suchas those described herein, including without limitation, a secreted formof TRAIL or a functional domain thereof (e.g., a secreted form of theextracellular domain), multimodal TRAIL, or a therapeutic TRAIL module,therapeutic TRAIL domain (e.g., the extracellular domain) or therapeuticTRAIL variant (examples of each of which are described inWO2012/106281), and also fragments, variants and derivatives of these,and fusion proteins comprising one of these TRAIL forms such asdescribed herein.

TRAIL is normally expressed on both normal and tumor cells as a noncovalent homotrimeric type-II transmembrane protein (memTRAIL). Inaddition, a naturally occurring soluble form of TRAIL (solTRAIL) can begenerated due to alternative mRNA splicing or proteolytic cleavage ofthe extracellular domain of memTRAIL and thereby still retainingtumor-selective pro-apoptotic activity. TRAIL utilizes an intricatereceptor system comprising four distinct membrane receptors, designatedTRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4. Of these receptors, onlyTRAIL-R1 and TRAIL-2 transmit an apoptotic signal. These two receptorsbelong to a subgroup of the TNF receptor family, the so-called deathreceptors (DRs), and contain the hallmark intracellular death domain(DD). This DD is critical for apoptotic signaling by death receptors (J.M. A. Kuijlen et al., Neuropathology and Applied Neurobiology, 2010 Vol.36 (3), pp. 168-182).

Apoptosis is integral to normal, physiologic processes that regulatecell number and results in the removal of unnecessary or damaged cells.Apoptosis is frequently dysregulated in human cancers, and recentadvancements in the understanding of the regulation of programmed celldeath pathways has led to the development of agents to reactivate oractivate apoptosis in malignant cells. This evolutionarily conservedpathway can be triggered in response to damage to key intracellularstructures or the presence or absence of extracellular signals thatprovide normal cells within a multicellular organism with contextualinformation.

Without meaning to be bound by theory, TRAIL activates the “extrinsicpathway” of apoptosis by binding to TRAIL-R1 and/or TRAIL-R2, whereuponthe adaptor protein Fas-associated death domain and initiator caspase-8are recruited to the DD of these receptors. Assembly of this“death-inducing signaling complex” (DISC) leads to the sequentialactivation of initiator and effector caspases, and ultimately results inapoptotic cell death. The extrinsic apoptosis pathway triggers apoptosisindependently of p53 in response to pro-apoptotic ligands, such asTRAIL. TRAIL-R1 can induce apoptosis after binding non-cross-linked andcross-linked sTRAIL. TRAIL-R2 can only be activated by cross-linkedsTRAIL. Death receptor binding leads to the recruitment of the adaptorFADD and initiator procaspase-8 and 10 to rapidly form the DISC.Procaspase-8 and 10 are cleaved into its activated configurationcaspase-8 and 10. Caspase-8 and 10 in turn activate the effectorcaspase-3, 6 and 7, thus triggering apoptosis.

In certain cells, the execution of apoptosis by TRAIL further relies onan amplification loop via the “intrinsic mitochondrial pathway” ofapoptosis. The mitochondrial pathway of apoptosis is a stress-activatedpathway, e.g., upon radiation, and hinges on the depolarization of themitochondria, leading to release of a variety of pro-apoptotic factorsinto the cytosol. Ultimately, this also triggers effector caspaseactivation and apoptotic cell death. This mitochondrial release ofpro-apoptotic factors is tightly controlled by the Bcl-2 family of pro-and anti-apoptotic proteins. In the case of TRAIL receptor signaling,the Bcl-2 homology (BH3) only protein ‘Bid’ is cleaved into a truncatedform (tBid) by active caspase-8. Truncated Bid subsequently activatesthe mitochondrial pathway.

TRAIL-R3 is a glycosylphosphatidylinositol-linked receptor that lacks anintracellular domain, whereas TRAIL-R4 only has a truncated andnon-functional DD. The latter two receptors are thought, without wishingto be bound or limited by theory, to function as decoy receptors thatmodulate TRAIL sensitivity. Evidence suggests that TRAIL-R3 binds andsequesters TRAIL in lipid membrane microdomains. TRAIL-R4 appears toform heterotrimers with TRAIL-R2, whereby TRAIL-R2-mediated apoptoticsignaling is disrupted. TRAIL also interacts with the soluble proteinosteoprotegerin

Diffuse expression of TRAIL has been detected on liver cells, bileducts, convoluted tubules of the kidney, cardiomyocytes, lung epithelia,Leydig cells, normal odontogenic epithelium, megakaryocytic cells anderythroid cells. In contrast, none or weak expression of TRAIL wasobserved in colon, glomeruli, Henle's loop, germ and Sertoli cells ofthe testis, endothelia in several organs, smooth muscle cells in lung,spleen and in follicular cells in the thyroid gland. TRAIL proteinexpression was demonstrated in glial cells of the cerebellum in onestudy. Vascular brain endothelium appears to be negative for TRAIL-R1and weakly positive for TRAIL-R2. With regard to the decoy receptors,TRAIL-R4 and TRAIL-R3 have been detected on oligodendrocytes andneurones.

TRAIL-R1 and TRAIL-R2 are ubiquitously expressed on a variety of tumortypes. In a study on 62 primary GBM tumor specimens, TRAIL-R1 andTRAIL-R2 were expressed in 75% and 95% of the tumors, respectively. Ofnote, a statistically significant positive association was identifiedbetween agonistic TRAIL receptor expression and survival. Highlymalignant tumors express a higher amount of TRAIL receptors incomparison with less malignant tumors or normal tissue. In generalTRAIL-R2 is more frequently expressed on tumor cells than TRAIL-R1.

“Tumor necrosis factor-related apoptosis-inducing ligand” or “TRAIL” asused herein refers to the 281 amino acid polypeptide having the aminoacid sequence of:MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO: 1), asdescribed by, e.g., NP_003801.1, together with any naturally occurringallelic, splice variants, and processed forms thereof. Typically, TRAILrefers to human TRAIL. The term TRAIL, in some embodiments of theaspects described herein, is also used to refer to truncated forms orfragments of the TRAIL polypeptide, comprising, for example, specificTRAIL domains or residues thereof. The amino acid sequence of the humanTRAIL molecule as presented in SEQ ID NO: 1 comprises an N-terminalcytoplasmic domain (amino acids 1-18), a transmembrane region (aminoacids 19-38), and an extracellular domain (amino acids 39-281). Theextracellular domain comprises the TRAIL receptor-binding region. TRAILalso has a spacer region between the C-terminus of the transmembranedomain and a portion of the extracellular domain This spacer region,located at the N-terminus of the extracellular domain, consists of aminoacids 39 through 94 of SEQ ID NO: 1. Amino acids 138 through 153 of SEQID NO: 1 correspond to a loop between the sheets of the folded (threedimensional) human TRAIL protein.

In one embodiment, the TRAIL comprises the extracellular domain of TRAIL(e.g., human trial). In one embodiment, the TRAIL is a fusion proteincomprising one or more domains of TRAIL (e.g., the extracellular domain)fused to a heterologous sequence. In one embodiment, the TRAIL fusionprotein further comprises a signal for secretion.

Preferably, the TRAIL protein and the nucleic acids encoding it, arederived from the same species as will be administered in the therapeuticmethods described herein. In one embodiment, the nucleotide sequenceencoding TRAIL and the TRAIL amino acid sequence is derived from amammal. In one embodiment, the mammal is a human (human TRAIL). In oneembodiment, the mammal is a non-human primate.

Fragments, Variants and Derivatives of TRAIL

Fragments, variants and derivatives of native TRAIL proteins for use inthe invention that retain a desired biological activity of TRAIL, suchas TRAIL apoptotic activity are also envisioned for delivery by theoncolytic virus vector. In one embodiment, the biological or apoptoticactivity of a fragment, variant or derivative of TRAIL is essentiallyequivalent to the biological activity of the corresponding native TRAILprotein. In one embodiment, the biological activity for use indetermining the activity is apoptotic activity. In one embodiment, 100%of the apoptotic activity is retained by the fragment, variant orderivative. In one embodiment less than 100%, activity is retained(e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%) ascompared to the full length native TRAIL. Fragments, variants orderivatives which retain less activity (e.g., 34%, 30%, 25%, 20%, 10%,etc.) may also be of value in the therapeutic methods described hereinand as such are also encompassed in the invention. One measurement ofTRAIL apoptotic activity by a TRAIL variant or TRAIL domain is theability to induce apoptotic death of Jurkat cells. Assay procedures foridentifying biological activity of TRAIL variants by detecting apoptosisof target cells, such as Jurkat cells, are well known in the art. DNAladdering is among the characteristics of cell death via apoptosis, andis recognized as one of the observable phenomena that distinguishapoptotic cell death from necrotic cell death. Apoptotic cells can alsobe identified using markers specific for apoptotic cells, such asAnnexin V, in combination with flow cytometric techniques, as known toone of skill in the art. Further examples of assay techniques suitablefor detecting death or apoptosis of target cells include those describedin WO2012/106281.

A variety of TRAIL fragments that retain the apoptotic activity of TRAILare known in the art, and include biologically active domains andfragments disclosed in Wiley et al. (U.S. Patent Publication20100323399).

TRAIL variants can be obtained by mutations of native TRAIL nucleotidesequences, for example. A “TRAIL variant,” as referred to herein, is apolypeptide substantially homologous to a native TRAIL, but which has anamino acid sequence different from that of native TRAIL because of oneor a plurality of deletions, insertions or substitutions. “TRAILencoding DNA sequences” encompass sequences that comprise one or moreadditions, deletions, or substitutions of nucleotides when compared to anative TRAIL DNA sequence, but that encode a TRAIL protein or fragmentthereof that is essentially biologically equivalent to a native TRAILprotein, i.e., has the same apoptosis inducing activity.

The variant amino acid or DNA sequence preferably is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, identical to a nativeTRAIL sequence. The degree of homology or percent identity) between anative and a mutant sequence can be determined, for example, bycomparing the two sequences using freely available computer programscommonly employed for this purpose on the world wide web.

Alterations of the native amino acid sequence can be accomplished by anyof a number of known techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smithet al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties.

TRAIL variants can, in some embodiments, comprise conservativelysubstituted sequences, meaning that one or more amino acid residues of anative TRAIL polypeptide are replaced by different residues, and thatthe conservatively substituted TRAIL polypeptide retains a desiredbiological activity, i.e., apoptosis inducing activity or TRAILapoptotic activity, that is essentially equivalent to that of the nativeTRAIL polypeptide. Examples of conservative substitutions includesubstitution of amino acids that do not alter the secondary and/ortertiary structure of TRAIL.

In other embodiments, TRAIL variants can comprise substitution of aminoacids that have not been evolutionarily conserved. Conserved amino acidslocated in the C-terminal portion of proteins in the TNF family, andbelieved to be important for biological activity, have been identified.These conserved sequences are discussed in Smith et al. (Cell, 73:1349,1993); Suda et al. (Cell, 75:1169, 1993); Smith et al. (Cell, 76:959,1994); and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993).Advantageously, in some embodiments, these conserved amino acids are notaltered when generating conservatively substituted sequences. In someembodiments, if altered, amino acids found at equivalent positions inother members of the TNF family are substituted. Among the amino acidsin the human TRAIL protein of SEQ ID NO:1 that are conserved are thoseat positions 124-125 (AH), 136 (L), 154 (W), 169 (L), 174 (L), 180 (G),182 (Y), 187 (Q), 190 (F), 193 (Q), and 275-276 (FG) of SEQ ID NO:1.Another structural feature of TRAIL is a spacer region (i.e., TRAIL(39-94)) between the C-terminus of the transmembrane region and theportion of the extracellular domain that is believed to be important forbiological apoptotic activity. In some embodiments, when the desiredbiological activity of TRAIL domain is the ability to bind to a receptoron target cells and induce apoptosis of the target cells substitution ofamino acids occurs outside of the receptor-binding domain.

A given amino acid of a TRAIL domain can, in some embodiments, bereplaced by a residue having similar physiochemical characteristics,e.g., substituting one aliphatic residue for another (such as Ile, Val,Leu, or Ala for one another), or substitution of one polar residue foranother (such as between Lys and Arg; Glu and Asp; or Gln and Asn).Other such conservative substitutions, e.g., substitutions of entireregions having similar hydrophobicity characteristics, are well known.TRAIL polypeptides comprising conservative amino acid substitutions canbe tested in any one of the assays described herein to confirm that adesired TRAIL apoptotic activity of a native TRAIL molecule is retained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H).

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Particularly preferred conservative substitutions for use in the TRAILvariants described herein are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln;Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, intoGln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

Any cysteine residue not involved in maintaining the proper conformationof the multimodal TRAIL agent also can be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) can be added to themultimodal TRAIL agent to improve its stability or facilitateoligomerization.

Secreted TRAIL

In one embodiment, a form of TRAIL that is secreted (secreted TRAIL orsol TRAIL) is expressed by the oncolytic virus described herein. Variousforms of secreted TRAIL can be used in the methods and compositionsdescribed herein. In one embodiment, the secreted TRAIL is the naturallyoccurring soluble TRAIL. (Ashkenazi A. et al., J Clin Oncol 2008; 26:3621-30, and Kelley S K et al., J Pharmacol Exp Ther 2001; 299: 31-8).In one embodiment the naturally occurring soluble TRAIL is fused with anantibody derivative, such as scFv245 (Bremer E. et al., J Mol Med 2008;86: 909-24; Bremer E, et al., Cancer Res 2005; 65: 3380-88; Bremer E, etal., J Biol Chem 2005; 280: 10025-33, and Stieglmaier J, et al., CancerImmunol Immunother 2008; 57: 233-46).

Alternatively, the endogenous secretion sequence of TRAIL present on theN terminus can be replaced with the signal sequence (otherwise referredto as the extracellular domain) from Flt3 ligand and an isoleucinezipper (Shah et al., Cancer Research 64: 3236-3242 (2004); WO2012/106281; Shah et al. Mol Ther. 2005 June; 11(6):926-31). Othersecretion signal sequences can be added to TRAIL in turn to generate asecreted TRAIL for use in the invention. For example, SEC2 signalsequence and SEC(CV) signal sequence can be fused to TRAIL (see forexample U.S. Patent Publication 2002/0128438). Other secretion signalsequences may also be used and nucleotides including restriction enzymesites can be added to the 5′ or 3′ terminal of respective secretionsignal sequence, to facilitate the incorporation of such sequences intothe DNA cassette. Such secretion signal sequences can be fused to theN-terminus or to the C-terminus.

Additionally, a linker sequence may be inserted between heterologoussequence and the TRAIL in order to preserve function of either portionof the generated fusion protein. Such linker sequences known in the artinclude a linker domain having the 7 amino acids (EASGGPE; SEQ ID NO:3), a linker domain having 18 amino acids (GSTGGSGKPGSGEGSTGG; SEQ IDNO: 4). As used herein, a “linker sequence” refers to a peptide, or anucleotide sequence encoding such a peptide, of at least 8 amino acidsin length. In some embodiments of the aspects described herein, thelinker module comprises at least 9 amino acids, at least 10 amino acids,at least 11 amino acids, at least 12 amino acids, at least 13 aminoacids, at least 14 amino acids, at least 15 amino acids, at least 16amino acids, at least 17 amino acids, at least 18 amino acids, at least19 amino acids, at least 20 amino acids, at least 21 amino acids, atleast 22 amino acids, at least 23 amino acids, at least 24 amino acids,at least 25 amino acids, at least 30 amino acids, at least 35 aminoacids, at least 40 amino acids, at least 45 amino acids, at least 50amino acids, at least 55 amino acids, at least 56 amino acids, at least60 amino acids, or least 65 amino acids. In some embodiments of theaspects described herein, a linker module comprises a peptide of 18amino acids in length. In some embodiments of the aspects describedherein, a linker module comprises a peptide of at least 8 amino acids inlength but less than or equal to 56 amino acids in length, i.e., thelength of the spacer sequence in the native TRAIL molecule of SEQ IDNO: 1. In some embodiments, the linker sequence comprises the spacersequence of human TRAIL, i.e., amino acids 39-94 of SEQ ID NO: 1, or asequence having at least 80%, at least 85%, at least 90%, at least 95%,at least 99% identity to amino acids 39-94 of SEQ ID NO: 1.

Signal Sequences

Secreted TRAIL may be generated by incorporation of a secretion signalsequence into the TRAIL or TRAIL fragment or derivative. As used herein,the terms “secretion signal sequence,” “secretion sequence,” “secretionsignal peptide,” or “signal sequence,” refer to a sequence that isusually about 3-60 amino acids long and that directs the transport of apropeptide to the endoplasmic reticulum and through the secretorypathway during protein translation. As used herein, a signal sequence,which can also be known as a signal peptide, a leader sequence, a preprosequence or a pre sequence, does not refer to a sequence that targets aprotein to the nucleus or other organelles, such as mitochondria,chloroplasts and apicoplasts. In one embodiment, the secretion signalsequence comprises 5 to 15 amino acids with hydrophobic side chains thatare recognized by a cytosolic protein, SRP (Signal RecognitionParticle), which stops translation and aids in the transport of anmRNA-ribosome complex to a translocon in the membrane of the endoplasmicreticulum. In one embodiment, the secretion signal peptide comprises atleast three regions: an amino-terminal polar region (N region), wherefrequently positive charged amino acid residues are observed, a centralhydrophobic region (H region) of 7-8 amino acid residues and acarboxy-terminal region (C region) that includes the cleavage site.Commonly, the signal peptide is cleaved from the mature protein withcleavage occurring at this cleavage site.

The secretory signal sequence is operably linked to the TRAIL or TRAILfragment or derivative such that the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the nucleotide sequenceencoding the polypeptide of interest, although certain secretory signalsequences can be positioned elsewhere in the nucleotide sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830).

In one embodiment, the secretory sequence comprises amino acids 1-81 ofthe following Flt3L amino acid sequence: MTVLAPAWSP NSSLLLLLLLLSPCLRGTPD CYFSHSPISS NFKVKFRELT DHLLKDYPVT VAVNLQDEKH CKALWSLFLAQRWIEQLKTV AGSKMQTLLE DVNTEIHFVT SCTFQPLPEC LRFVQTNISH LLKDTCTQLLALKPCIGKAC QNFSRCLEVQ CQPDSSTLLP PRSPIALEAT ELPEPRPRQL LLLLLLLLPLTLVLLAAAWG LRWQRARRRG ELHPGVPLPS HP (SEQ ID NO: 2, GenBank AccessionP49772), or a functional fragment thereof. In one embodiment, the signalpeptide comprises amino acids 1-81 of SEQ ID NO: 2. In one embodiment,the secretory signal sequence comprises a sequence having at least 90%identity to amino acids 1-81 of SEQ ID NO: 2. In one embodiment, thesecretory signal sequence consists essentially of amino acids 1-81 ofSEQ ID NO: 2. In one embodiment, the secretory signal sequence consistsof amino acids 1-81 of SEQ ID NO: 2.

While the secretory signal sequence can be derived from Flt3L, in otherembodiments a suitable signal sequence can also be derived from anothersecreted protein or synthesized de novo. Other secretory signalsequences which can be substituted for the Flt3L signal sequence forexpression in eukaryotic cells include, for example, naturally-occurringor modified versions of the human IL-17RC signal sequence, otPA pre-prosignal sequence, human growth hormone signal sequence, human CD33 signalsequence Ecdysteroid Glucosyltransferase (EGT) signal sequence, honeybee Melittin (Invitrogen Corporation; Carlsbad, Calif.), baculovirusgp67 (PharMingen: San Diego, Calif.) (US Pub. No. 20110014656).Additional secretory sequences include secreted alkaline phosphatasesignal sequence, interleukin-1 signal sequence, CD-14 signal sequenceand variants thereof (US Pub. No. 20100305002) as well as the followingpeptides and derivatives thereof: Sandfly Yellow related protein signalpeptide, silkworm friboin LC signal peptide, snake PLA2, Cyrpidinanoctiluca luciferase signal peptide, and pinemoth fibroin LC signalpeptide (US Pub. No. 20100240097). Further signal sequences can beselected from databases of protein domains, such as SPdb, a signalpeptide database described in Choo et al., BMC Bioinformatics 2005,6:249, LOCATE, a mammalian protein localization database described inSprenger et al. Nuc Acids Res, 2008, 36:D230D233, or identified usingcomputer modeling by those skilled in the art (Ladunga, Curr OpinBiotech 2000, 1:13-18).

Selection of appropriate signal sequences and optimization orengineering of signal sequences is known to those skilled in the art(Stern et al., Trends in Cell & Molecular Biology 2007 2:1-17; Barash etal., Biochem Biophys Res Comm 2002, 294:835-842). In one embodiment, asignal sequence can be used that comprise a protease cleavage site for asite-specific protease (e.g., Factor IX or Enterokinase). This cleavagesite can be included between the pro sequence and the bioactive secretedpeptide sequence, e.g., TRAIL domain, and the pro-peptide can beactivated by the treatment of cells with the site-specific protease (USPub. No. 20100305002).

Leucine Zippers

The TRAIL or TRAIL fragment, derivative or variant, described hereincan, in some embodiments, further comprise a leucine zipper domainsequence. As used herein, “leucine zipper domains” refer to naturallyoccurring or synthetic peptides that promote oligomerization of theproteins in which they are found. The leucine zipper is asuper-secondary structure that functions as a dimerization domain, andits presence generates adhesion forces in parallel alpha helices. Asingle leucine zipper comprises multiple leucine residues atapproximately 7-residue intervals, which forms an amphipathic alphahelix with a hydrophobic region running along one side. The dimer formedby a zipper domain is stabilized by the heptan repeat, designated(abcdefg)_(n) according to the notation of McLachlan and Stewart (J.Mol. Biol. 98:293; 1975), in which residues a and d are generallyhydrophobic residues, with d being a leucine, which line up on the sameface of a helix. Oppositely-charged residues commonly occur at positionsg and e. Thus, in a parallel coiled coil formed from two helical zipperdomains, the “knobs” formed by the hydrophobic side chains of the firsthelix are packed into the “holes” formed between the side chains of thesecond helix. The residues at position d (often leucine) contributelarge hydrophobic stabilization energies, and are important for oligomerformation (Krystek et al., Int. J. Peptide Res. 38:229, 1991). Thishydrophobic region provides an area for dimerization, allowing themotifs to “zip” together. Furthermore, the hydrophobic leucine region isabsolutely required for DNA binding. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize.

Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). The nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The fusogenic proteins of several differentviruses, including paramyxovirus, coronavirus, measles virus and manyretroviruses, also possess zipper domains (Buckland and Wild, Nature338:547, 1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDSResearch and Human Retrovirtises 6:703, 1990). The zipper domains inthese fusogenic viral proteins are near the transmembrane region of theprotein. Oligomerization of fusogenic viral proteins is involved infusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A.88:3523, 1991). Zipper domains have also been reported to play a role inoligomerization of heat-shock transcription factors (Rabindran et al.,Science 259:230, 1993).

Examples of leucine zipper domains suitable for producing multimodalTRAIL agents include, but are not limited to, those described in PCTapplication WO 94/10308; U.S. Pat. No. 5,716,805; the leucine zipperderived from lung surfactant protein D (SPD) described in Hoppe et al.,1994, FEBS Letters 344:191; and Fanslow et al., 1994, Semin. Immunol.6:267-278, the contents of each of which are hereby incorporated byreference in their entireties. In one embodiment, leucine residues in aleucine zipper domain are replaced by isoleucine residues. Such peptidescomprising isoleucine can also be referred to as isoleucine zippers, butare encompassed by the term “leucine zippers” as used herein.

Additional Nucleic Acids

The recombinant oncolytic virus comprising TRAIL nucleic acid mayfurther contain additional heterologous nucleic acid sequences (e.g., inexpressible form), referred to herein as a second heterologous nucleicacid sequence, a third heterologous nucleic acid sequence, etc.Alternatively, the recombinant oncolytic virus may contain no additionalheterologous nucleic acid sequences.

Any desired DNA can be inserted, including DNA that encodes selectablemarkers, or preferably genes coding for a therapeutic, biologicallyactive protein, such as interferons, cytokines, chemokines, or morepreferably DNA coding for a prodrug converting enzyme, includingthymidine kinase (Martuza et al., Science, 252:854, 1991), cytosinedeamindase (U.S. Pat. No. 5,358,866), cyp450 (U.S. Pat. No. 5,688,773),and others. In one embodiment, the nucleic acid encodes a protein thatinhibits tumor growth (e.g., a chemotherapeutic, growth regulatoryagent) or modifies an immune response. An example of a chemotherapeuticagent is mitomicin C. In one embodiment, the nucleic acid encodes agrowth regulatory molecule (e.g., one that has been lost intumorigenesis of the tumor). Examples of such molecules withoutlimitation proteins from the caspase family such as Caspase-9(P55211(CASP9_HUMAN); HGNC: 15111; Entrez Gene: 8422; Ensembl:ENSG000001329067; OMIM: 6022345; UniProtKB: P552113), Caspase-8 (Q14790(CASP8_HUMAN); 9606 [NCBI]), Caspase-7 (P55210 (CASP7_HUMAN); 9606[NCBI]), and Caspase-3 (HCGN: 1504; Ensembl:ENSG00000164305; HPRD:02799;MIM:600636; Vega:OTTHUMG00000133681), pro-apoptotic proteins such as Bax(HGNC: 9591; Entrez Gene: 5812; Ensembl: ENSG000000870887; OMIM:6000405; UniProtKB: Q078123), Bid (HGNC: 10501; Entrez Gene: 6372;Ensembl: ENSG000000154757; OMIM: 6019975; UniProtKB: P559573), Bad(HGNC: 9361; Entrez Gene: 5722; Ensembl: ENSG000000023307; OMIM:6031675; UniProtKB: Q92934), Bak (HGNC: 9491; Entrez Gene: 5782;Ensembl: ENSG000000301107; OMIM: 6005165; UniProtKB: Q166113), BCL2L11(HGNC: 9941; Entrez Gene: 100182; Ensembl: ENSG000001530947; OMIM:6038275; UniProtKB: 0435213), p53 (HGNC: 119981; Entrez Gene: 71572;Ensembl: ENSG000001415107; OMIM: 1911705; UniProtKB: P046373), PUMA(HGNC: 178681; Entrez Gene: 271132; Ensembl: ENSG000001053277; OMIM:6058545; UniProtKB: Q96PG83; UniProtKB: Q9BXH13), Diablo/SMAC (HGNC:215281; Entrez Gene: 566162; Ensembl: ENSG000001840477; OMIM: 6052195;UniProtKB: Q9NR283). In one embodiment, the nucleic acid encodes animmunomodulatory agent (e.g, immunostimulatory transgenes), including,without limitation, Flt-3 ligand, HMBG1, calreticulin, GITR ligand,interleukin-12, interleukin-15, interleukin-18, or CCL17.

The exogenous nucleic acids can be inserted into the oncolytic virus bythe skilled practitioner. In one embodiment, the oncolytic virus is HSVand the exogenous nucleic acid is inserted into the thymidine kinase(TK) gene of the viral genome, or replacing the deleted TK gene (see forexample, U.S. Pat. No. 5,288,641 for insertion of exogenous nucleic acidinto HSV). When the oncolytic virus comprises a second exogenous nucleicacid, the nucleic acid preferably encodes an anti-oncogenic or oncolyticgene product. The gene product may be one (e.g. an antisenseoligonucleotide) which inhibits growth or replication of only the cellinfected by the virus, or it may be one (e.g. thymidine kinase) whichexerts a significant bystander effect upon lysis of the cell infected bythe virus.

Methods of Treatment

Another aspect of the invention relates to a method of treating aproliferative disorder in a subject. The method comprises administeringa recombinant oncolytic virus comprising the TRAIL nucleic acidsequences (herein referred to as recombinant oncolytic virus-TRAIL)described herein to the subject to thereby contact cells exhibitingundesired proliferation with an effective amount of the recombinantoncolytic virus-TRAIL. In one embodiment, the subject is diagnosed witha tumor that is resistant to an oncolytic virus (e.g, oHSV), to TRAIL orsecreted TRAIL, or a combination thereof.

In one embodiment, the proliferative disorder is a tumor and the methodof the invention relates to a method for inhibiting tumor progression.An effective amount of the recombinant oncolytic virus-TRAIL iscontacted to the tumor to thereby deliver the molecule to the tumorcells.

The term “tumor” refers to the tissue mass or tissue type or cell typethat is undergoing uncontrolled proliferation. A tumor can be benign ormalignant. A benign tumor is characterized as not undergoing metastasis.A malignant cell is a cancer cell and can undergo metastasis. Tumors onwhich the method can be performed include, without limitation, adenoma,angio-sarcoma, astrocytoma, epithelial carcinoma, germinoma,glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma,hematoma, hepato-blastoma, leukemia, lymphoma, medulloblastoma,melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma,sarcoma, and teratoma. The tumor can be chosen from acral lentiginousmelanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma,adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors,bartholin gland carcinoma, basal cell carcinoma, bronchial glandcarcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous,cholangio-carcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrialhyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma,ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodularhyperplasia, gastrinoma, germ cell tumors, glioblastoma, glucagonoma,hemangiblastomas, hemangioendothelioma, hemangiomas, hepatic adenoma,hepatic adenomatosis, hepatocellular carcinoma, insulinoma,intaepithelial neoplasia, interepithelial squamous cell neoplasia,invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,lentigo maligna melanomas, malignant melanoma, malignant mesothelialtumors, medulloblastoma, medulloepithelioma, melanoma, meningeal,mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat cellcarcinoma, oligodendroglial, osteosarcoma, pancreatic, papillary serousadeno-carcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudo-sarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,verrucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor. In one embodiment, the tumor expresses one or more receptors forTRAIL.

Another aspect of the invention relates to a method of treating asubject for a cell proliferative disorder such as cancer. The methodcomprises administering a therapeutically effective amount ofrecombinant oncolytic virus-TRAIL to the subject in the form of apharmaceutical composition. Examples of cancer include but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include, but are not limited to,basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer;brain and CNS cancer; breast cancer; cancer of the peritoneum; cervicalcancer; choriocarcinoma; colon and rectum cancer; connective tissuecancer; cancer of the digestive system; endometrial cancer; esophagealcancer; eye cancer; cancer of the head and neck; gastric cancer(including gastrointestinal cancer); glioblastoma (GBM); hepaticcarcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer;larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung); lymphoma including Hodgkin's andnon-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavitycancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer;pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; salivary glandcarcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer;testicular cancer; thyroid cancer; uterine or endometrial cancer; cancerof the urinary system; vulval cancer; as well as other carcinomas andsarcomas; as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

In one embodiment the cancer is a brain cancer, brain tumor, orintracranial neoplasm. Intracranial neoplasms or cancers can arise fromany of the structures or cell types present in the CNS, including thebrain, meninges, pituitary gland, skull, and even residual embryonictissue. The overall annual incidence of primary brain tumors in theUnited States is 14 cases per 100,000. The most common primary braintumors are meningiomas, representing 27% of all primary brain tumors,and glioblastomas, representing 23% of all primary brain tumors (whereasglioblastomas account for 40% of malignant brain tumor in adults). Manyof these tumors are aggressive and of high grade. Primary brain tumorsare the most common solid tumors in children and the second mostfrequent cause of cancer death after leukemia in children.

Pharmaceutical Compositions

The pharmaceutically acceptable vehicle for delivery of the recombinantoncolytic virus-TRAIL can be selected from known pharmaceuticallyacceptable vehicles, and should be one in which the virus is stable. Forexample, it can be a diluent, solvent, buffer, and/or preservative. Anexample of a pharmaceutically acceptable vehicle is phosphate buffercontaining NaCl. Other pharmaceutically acceptable vehicles aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15thEd. Easton: Mack Publishing Co. pp 1405-1412 and 1461-1487 (1975) andTHE NATIONAL FORMULARY XIV., 14th Ed. Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference.

Pharmaceutical compositions and formulations for specified modes ofadministration, described herein are also encompassed by the presentinvention. In one embodiment, the oncolytic virus-TRAIL described hereinis an active ingredient in a composition comprising a pharmaceuticallyacceptable carrier. Such a composition is referred to herein as apharmaceutical composition. A “pharmaceutically acceptable carrier”means any pharmaceutically acceptable means to mix and/or deliver thetargeted delivery composition to a subject. The term “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting the subject agents from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and is compatible with administration toa subject, for example a human. Such compositions can be specificallyformulated for administration via one or more of a number of routes,such as the routes of administration described herein. Supplementaryactive ingredients also can be incorporated into the compositions. Whenan agent, formulation or pharmaceutical composition described herein, isadministered to a subject, preferably, a therapeutically effectiveamount is administered. As used herein, the term “therapeuticallyeffective amount” refers to an amount that results in an improvement orremediation of the condition.

Administration

Administration of the pharmaceutical composition to a subject is bymeans which the recombinant oncolytic virus-TRAIL contained therein willcontact the target cell. The specific route will depend upon certainvariables such as the target cell, and can be determined by the skilledpractitioner. Suitable methods of administering a composition comprisinga pharmaceutical composition of the present invention to a patientinclude any route of in vivo administration that is suitable fordelivering a viral vector, recombinant nucleic acid molecule or proteinto a patient. The preferred routes of administration will be apparent tothose of skill in the art, depending on the type of viral vector used,the target cell population, and the disease or condition experienced bythe subject. Preferred methods of in vivo administration include, butare not limited to, intravenous administration, intraperitonealadministration, intramuscular administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation (e.g., aerosol), intracerebral, nasal, oral, pulmonaryadministration, impregnation of a catheter, and direct injection into atissue. In an embodiment where the target cells are in or near a tumor,a preferred route of administration is by direct injection into thetumor or tissue surrounding the tumor. For example, when the tumor is abreast tumor, the preferred methods of administration includeimpregnation of a catheter, and direct injection into the tumor.

Intravenous, intraperitoneal, and intramuscular administrations can beperformed using methods standard in the art. Aerosol (inhalation)delivery can also be performed using methods standard in the art (see,for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference in itsentirety). Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers, include plastic capsules or tablets, such asthose known in the art.

One method of local administration is by direct injection. Directinjection techniques are particularly useful for administering arecombinant nucleic acid molecule to a cell or tissue that is accessibleby surgery, and particularly, on or near the surface of the body.Administration of a composition locally within the area of a target cellrefers to injecting the composition centimeters and preferably,millimeters from the target cell or tissue.

Dosage and Treatment Regimen

The appropriate dosage and treatment regimen for the methods oftreatment described herein will vary with respect to the particulardisease being treated, the molecules being delivered, and the specificcondition of the subject. The skilled practitioner is to determine theamounts and frequency of administration on a case by case basis. In oneembodiment, the administration is over a period of time until thedesired effect (e.g., reduction in symptoms is achieved). In oneembodiment, administration is 1, 2, 3, 4, 5, 6, or 7 times per week. Inone embodiment, administration is over a period of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 weeks. In one embodiment, administration is over a period of2, 3, 4, 5, 6 or more months. In one embodiment, treatment is resumedfollowing a period of remission.

Kits

Another aspect of the invention relates to a kit comprising one or moreof the compositions described herein. Optionally the kit can include thecompositions distributed in single or multiple dosages units. The kitmay comprise the composition packaged in a device for administration.The kit may optionally comprise directions for use, dosage, and/oradministration of the composition contained within.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A recombinant oncolytic virus comprising a nucleic acid sequence    encoding tumor necrosis factor-related apoptosis-inducing ligand    (TRAIL) or a biologically active fragment thereof, in expressible    form.-   2. The recombinant oncolytic virus of paragraph 1, wherein the    oncolytic virus is an oncolytic herpes simplex virus (oHSV).-   3. The recombinant oncolytic virus of paragraph 2, wherein the oHSV    is selected from the group consisting of G207, G47Δ HSV-R3616, 1716,    R3616, and R4009.-   4. The recombinant oncolytic virus of any one of paragraphs 1-3,    wherein the TRAIL is a secreted form of TRAIL (S-TRAIL).-   5. The recombinant oncolytic virus of any one of paragraphs 1-3,    wherein the TRAIL is a TRAIL fusion protein.-   6. The recombinant oncolytic virus of any one of paragraphs 2-5,    wherein the TRAIL is regulated by the HSV immediate early 4/5    promoter.-   7. The recombinant oncolytic virus of any one of paragraphs 1-6,    wherein the virus contains an additional exogenous nucleic acid in    expressible form.-   8. The recombinant oncolytic virus of any one of paragraphs 1-6    wherein the virus contains no additional exogenous nucleic acids.-   9. A nucleic acid comprising the genome of a recombinant oncolytic    virus of any one of paragraphs 1-8.-   10. A host cell comprising an oncolytic virus of any one of    paragraphs 1-8 or the nucleic acid of paragraph 9.-   11. The nucleic acid of paragraph 9 that is selected from the group    consisting of a bacterial artificial chromosome (BAC), a P1-derived    artificial chromosome (PAC), a yeast artificial chromosome (YAC) and    a human artificial chromosome (HAC).-   12. A pharmaceutical composition comprising a recombinant oncolytic    virus of any one of paragraphs 1-8.-   13. A kit comprising the pharmaceutical composition of paragraph 12    and instructions for use.-   14. A method of inhibiting tumor progression in a subject comprising    contacting the tumor with an effective amount of a recombinant    oncolytic virus of any one of paragraphs 1-8.-   15. The method of paragraph 14, wherein the tumor is a brain tumor.-   16. The method of paragraph 15, wherein the brain tumor is a glioma.-   17. The method of paragraph 14, wherein the tumor is malignant.-   18. The method of paragraph 17, wherein the tumor is selected from    the group consisting of adenoma, angio-sarcoma, astrocytoma,    epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma,    hemangioendothelioma, hemangiosarcoma, hematoma, hepato-blastoma,    leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma,    osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and    teratoma. The tumor can be chosen from acral lentiginous melanoma,    actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma,    adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors,    bartholin gland carcinoma, basal cell carcinoma, bronchial gland    carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma,    cavernous, cholangio-carcinoma, chondosarcoma, choriod plexus    papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal    sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma,    endometrioid adenocarcinoma, ependymal, epitheloid, Ewing's sarcoma,    fibrolamellar, focal nodular hyperplasia, gastrinoma, germ cell    tumors, glioblastoma, glucagonoma, hemangiblastomas,    hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic    adenomatosis, hepatocellular carcinoma, insulinoma, intaepithelial    neoplasia, interepithelial squamous cell neoplasia, invasive    squamous cell carcinoma, large cell carcinoma, leiomyosarcoma,    lentigo maligna melanomas, malignant melanoma, malignant mesothelial    tumors, medulloblastoma, medulloepithelioma, melanoma, meningeal,    mesothelial, metastatic carcinoma, mucoepidermoid carcinoma,    neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, oat    cell carcinoma, oligodendroglial, osteosarcoma, pancreatic,    papillary serous adeno-carcinoma, pineal cell, pituitary tumors,    plasmacytoma, pseudo-sarcoma, pulmonary blastoma, renal cell    carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous    carcinoma, small cell carcinoma, soft tissue carcinomas,    somatostatin-secreting tumor, squamous carcinoma, squamous cell    carcinoma, submesothelial, superficial spreading melanoma,    undifferentiated carcinoma, uveal melanoma, verrucous carcinoma,    vipoma, well differentiated carcinoma, and Wilm's tumor.-   19. The method of any one of paragraphs 14-18, wherein contacting is    by a method of administration to the subject is by a method selected    from the group consisting of intravenous administration,    intraperitoneal administration, intramuscular administration,    intracoronary administration, intraarterial administration,    subcutaneous administration, transdermal delivery, intratracheal    administration, subcutaneous administration, intraarticular    administration, intraventricular administration, inhalation,    intracerebral, nasal, oral, pulmonary administration, impregnation    of a catheter, and direct injection into a tissue or tumor.-   20. A method of treating cancer in a subject comprising    administering to the subject a therapeutically effective amount of    the pharmaceutical composition of paragraph 12, to thereby treat the    cancer.-   21. The method of paragraph 20, wherein the cancer is selected from    the group consisting of basal cell carcinoma, biliary tract cancer;    bladder cancer; bone cancer; brain and CNS cancer; breast cancer;    cancer of the peritoneum; cervical cancer; choriocarcinoma; colon    and rectum cancer; connective tissue cancer; cancer of the digestive    system; endometrial cancer; esophageal cancer; eye cancer; cancer of    the head and neck; gastric cancer (including gastrointestinal    cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma;    intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;    leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,    non-small cell lung cancer, adenocarcinoma of the lung, and squamous    carcinoma of the lung); lymphoma including Hodgkin's and    non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral    cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian    cancer; pancreatic cancer; prostate cancer; retinoblastoma;    rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;    salivary gland carcinoma; sarcoma; skin cancer; squamous cell    cancer; stomach cancer; testicular cancer; thyroid cancer; uterine    or endometrial cancer; cancer of the urinary system; vulval cancer;    as well as other carcinomas and sarcomas; as well as B-cell lymphoma    (including low grade/follicular non-Hodgkin's lymphoma (NHL); small    lymphocytic (SL) NHL; intermediate grade/follicular NHL;    intermediate grade diffuse NHL; high grade immunoblastic NHL; high    grade lymphoblastic NHL; high grade small non-cleaved cell NHL;    bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and    Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL);    acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic    myeloblastic leukemia; and post-transplant lymphoproliferative    disorder (PTLD), as well as abnormal vascular proliferation    associated with phakomatoses, edema, and Meigs' syndrome.-   22. The method of any one of paragraphs 20-21, wherein the cancer is    brain cancer.-   23. The method of paragraph 22, wherein the brain cancer is glioma    or glioblastoma.-   24. The method of any one of paragraphs 20-23, wherein    administration is by a method selected from the group consisting of    intravenous administration, intraperitoneal administration,    intramuscular administration, intracoronary administration,    intraarterial administration, subcutaneous administration,    transdermal delivery, intratracheal administration, subcutaneous    administration, intraarticular administration, intraventricular    administration, inhalation, intracerebral, nasal, oral, pulmonary    administration, impregnation of a catheter, and direct injection    into a tissue or tumor.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

In this study, a panel of established and patient derived primary GBMstem cell lines were screened for their sensitivity to a recombinantversion of G47Δ (referred to herein as oHSV) and TRAIL. In an effort todevelop anti-GBM therapies that target a broad spectrum of GBMs that areeither resistant to TRAIL-mediated apoptosis or resistant to bothoHSV-mediated oncolysis and TRAIL, oHSV-bearing secretable-TRAIL(oHSV-TRAIL) was engineered and a mechanism-based therapeutic approachto target resistant GBMs in vitro and in malignant and invasive GBMmodels in mice was extensively studied.

Results

Screening of different GBM lines reveals differential sensitivities tooHSV mediated oncolysis and S-TRAIL-mediated apoptosis. We screened acohort of both established GBM cell lines (Gli36, U87, U251, and LN229)and primary glioma stem cell (GSC) lines obtained from surgicalspecimens (GBM4, GBM6, BT74, and GBM8F) for their sensitivity topurified S-TRAIL- or oHSV-mediated cell death. While three establishedlines had varying sensitivity to TRAIL-induced apoptosis-mediated bycaspase-3 and -7, one established line (LN229) was fully resistant toTRAIL-mediated apoptosis. Among the primary GSC, GBM8F was fullyresistant to TRAIL whereas other GSC lines had varying sensitivity toTRAIL-induced apoptosis. Next, we evaluated the sensitivity ofestablished GBM lines and GSC lines to oHSV (G47Δ empty) mediatedoncolysis. Among the established lines, TRAIL resistant LN229 line wasalso resistant to oHSV-mediated oncolysis whereas all the GSC lines weresensitive to oHSV-mediated oncolysis. The amounts of virus released bythe oHSV-infected cells greatly varied among the GBM cell lines tested,and did not necessarily correspond to the sensitivity to oHSV-mediatedoncolysis. These results reveal the identification of GBM lines that areeither resistant to TRAIL-mediated apoptosis (LN229, GBM8F) or resistantto both oHSV-mediated oncolysis and TRAIL (LN229). Based on theseresults, we used LN229 and GBM8F GSC for further therapeutic evaluation(FIG. 1a ).

To evaluate oHSV replication and spread in oHSV and TRAIL resistant GBMcells and corresponding changes in cell viability, LN229 and GBM8F cellsengineered to express Renilla luciferase (Rluc)-mCherry (LN229-RmC,GBM8F-RmC) were infected with oHSV-bearing firefly luciferase(oHSV-Fluc, FIG. 8), and monitored by in vitro and in vivo dualbioluminescence imaging. In vitro, oHSV-Fluc replicates in both LN229and GBM8F cells as indicated by firefly luciferase (Fluc) expression,but oHSV replication was more robust and peaked earlier in GBM8F cellsthan LN229 cells (FIG. 1B and FIG. 9). Accordingly, while oHSV killedGBM8F cells efficiently (indicated by Rluc expression), LN229 cellsexhibited apparent resistance to the killing effect by oHSV (FIG. 1C).In vivo monitoring of virus infection demonstrated the ability ofintratumorally injected oHSV-Fluc to replicate in intracerebral tumorsgenerated with these GBM cells, and revealed patterns of virusreplication similar to the ones observed in vitro (FIGS. 1B and D).These data reveal that although oHSV replicates in both oHSV resistantand sensitive lines but results in killing only the sensitive GBM8F lineand suggest that GBMs might be heterogeneous in the responses to oHSV.

Combination of oHSV and S-TRAIL Leads to Apoptosis in TRAIL ResistantGBM Cells In Vitro

In order to evaluate the therapeutic potential of oHSV and S-TRAIL inresistant GBMs, established LN229 GBM cells and primary GBM8F (TRAILresistant) GBM cells were infected with oHSV and treated with purifiedS-TRAIL 6 hours post-infection. oHSV infection and subsequent S-TRAILtreatment resulted in significant decrease in viability of LN229 andGBM8F cells in culture, which was associated with increased caspase-3/7activation (FIGS. 2A-D). A significantly impaired activation ofcaspase-3/7 activity was observed when LN229 and GBM8F cells wereinfected with oHSV and treated with S-TRAIL in the presence ofpan-caspase inhibitor, Z-VAD-FMK (FIGS. 2A, B). This resulted incomplete reversal of both LN229 and GBM8F cell death in oHSV-infectedand S-TRAIL-treated cells (FIGS. 2C-D). Western blotting revealed thatcleavage of poly-ADP ribose polymerase (PARP), one of the maindownstream targets of caspase-3, was undetectable by either S-TRAIL oroHSV treatment in both LN229 and GBM8F cells, indicating thatoHSV-mediated cell death is primarily not dependent on caspase/PARPactivation, However, cleaved PARP was increased in cells treated withcombined oHSV and S-TRAIL (FIG. 2E, F). A significant reduction in PARPcleavage was observed when LN229 and GBM8F cells were infected with oHSVand treated with S-TRAIL in the presence of pan-caspase inhibitor,Z-VAD-FMK (FIG. 2E, F). These results revealed that oHSV and TRAILcombination leads to caspase-mediated apoptosis in TRAIL resistant andboth TRAIL and oHSV resistant GBM cells.

oHSV-TRAIL Targets Both Cell Proliferation and Death Pathways inResistant GBM Cells

In order to target both TRAIL resistant and oHSV resistant cell lines,we engineered an oHSV-bearing S-TRAIL (oHSV-TRAIL) using a BACcontaining the backbone structure of G47Δ, G47Δ-BAC^(25,26,27) (FIG. 8).The replicating ability of oHSV-TRAIL was similar to that of oHSV asrevealed by the virus yield quantified using plaque assay on Vero cells(FIG. 10A). The secretion of TRAIL in oHSV-TRAIL infected LN229 GBMcells was confirmed by ELISA (FIG. 10B). To evaluate the effect ofoHSV-TRAIL, LN229-RmC cells and GBM8F-RmC cells were infected with oHSVor oHSV-TRAIL at multiplicity of infection (MOI)=1 for 24, 48, 72, and96 hours. The changes in cell viability (Rluc activity) revealed thatoHSV-TRAIL infection resulted in significantly more potent cell killingin both LN229 and GBM8F cells as compared to oHSV infection (FIGS. 3Aand B and FIG. 10C, D). Annexin V staining analysis revealed that thenumber of apoptotic cells (Annexin V positive, propidium iodidenegative) was considerably increased in both LN229 and GBM8F cellpopulations post-oHSV-TRAIL infection (FIG. 10E). oHSV-TRAIL alsoresulted in significantly greater killing in oHSV and TRAIL sensitivehuman Gli36 GBM line as compared to oHSV infection (FIG. 11). Westernblot analysis of oHSV-TRAIL-infected LN229 and GBM8F cell lysates showedcleavage of caspases-8, -9 and PARP and no significant difference wasobserved in Bcl2 expression (FIG. 3C) and death receptor (DR)4/5expression (FIG. 12). These results reveal that oHSV-TRAIL infectioninduces caspase-mediated apoptosis in both TRAIL resistant LN229 andGBM8F glioma lines.

We next examined the mechanism of oHSV-TRAIL induced apoptosis inresistant GBM cells. As such, we assessed the activation of moleculesinvolved in the cell proliferation pathways, mitogen-activated proteinkinases (MAPKs) including extracellular signal-regulated protein kinase1 and 2 (ERK 1/2), c-Jun N-terminal kinase (JNK) and p38 in both LN229and GBM8F. A significantly impaired activation of ERK 1/2 was observedin LN229 cells infected with oHSV, which was further impaired byoHSV-TRAIL (FIG. 3C). There was a complete shutdown of ERK 1/2phosphorylation in both oHSV and oHSV-TRAIL infected GBM8F cells. JNKand p38 were activated in LN229 and GBM8F cells after oHSV andoHSV-TRAIL infection.

We next sought to determine the significance of the JNK/ERK MAP kinasesignaling alterations in oHSV-TRAIL-mediated cytotoxicity of both TRAILand oHSV resistant GBM cells. To examine the role of JNK upregulation,we treated resistant GBM cells with JNK inhibitor, SP600125. LN229 wereinfected with oHSV or oHSV-TRAIL in the absence and presence ofSP600125. Western blotting analysis showed SP600125 treatment resultedin the inhibition of JNK phosphorylation in oHSV and oHSV-TRAIL-infectedcells and also a significant reduction in the cleavage of PARP inoHSV-TRAIL-infected cells (FIG. 4A). Caspase-3/7 activity assays showeda significant decrease in the caspase-3/7 activation inoHSV-TRAIL-infected cells in the presence of SP600125 as compared to theinfected cells in the absence of SP600125 (FIG. 4B). We alsoinvestigated whether inhibition of ERK1/2 with MEK-ERK inhibitor, U0126,could mimic oHSV-mediated inhibition of ERK1/2 and sensitize TRAILresistant GBM cells to TRAIL-induced apoptosis. Western blottinganalysis showed that U0126 treatment of LN229 cells inhibited ERKphosphorylation and subsequent addition of purified S-TRAIL markedlyincreased PARP cleavage (FIG. 4C). Combined treatment with U0126 andS-TRAIL decreased cell viability in resistant LN229 GBM cells comparedwith single treatment (FIG. 4D). These results thus suggest thatoHSV-mediated downregulation of the ERK-MAPK and upregulation of JNKsignaling may contribute to apoptotic cell death in oHSV-TRAIL-infectedresistant GBM cells.

oHSV-TRAIL Inhibits GBM Growth and Invasion In Vivo and ProlongsSurvival of Mice Bearing Both TRAIL and oHSV Resistant GBM

We assessed the therapeutic efficacy of oHSV-TRAIL in intracranial GBMs,using LN229 and GBM8F glioma lines which were engineered to expressFluc-mCherry (LN229-FmC, GBM8F-FmC). Mice bearing establishedintracranial LN229-FmC GBMs were administered intratumorally with oHSV,oHSV-TRAIL, or phosphate-buffered saline (PBS) and followed for changesin tumor volumes by Fluc imaging. As expected, oHSV injection had noimpact on both tumor burden (FIG. 13A) and survival (FIG. 5A, PBS: 50.5days, oHSV: 49 days) in this resistant GBM model. In contrast,oHSV-TRAIL injection resulted in significant decrease in tumor volumescompared to both control (PBS) and oHSV treated mice (FIG. 13A) andprolonged survival of mice-bearing intracranial GBMs (FIG. 5A). Themedian survival of oHSV-TRAIL-treated mice was 69 days, which wassignificantly longer than PBS- and oHSV-treated mice (FIG. 5A; P=0.038oHSV and oHSV-TRAIL comparison, log-rank test). X-gal staining of thebrain sections collected 48 hours post oHSV or oHSV-TRAIL injectionshowed an extensive distribution of reporter β-galactosidase positivity,which overlapped with the tumor area, suggesting that the spread ofvirus infection was comparable between oHSV and oHSV-TRAIL (FIG. 5B).Immunohistochemical analysis and confocal microscopy showedsignificantly increased cleaved caspase-3 staining in sections fromoHSV-TRAIL-treated tumors as compared to oHSV and PBS treated tumors.Furthermore, no cleaved caspase-3 staining was seen in normal braincells in all treated groups revealing the tumor specificity ofoHSV-TRAIL treatment (FIG. 5C, D).

To examine the effect on GBM cell invasion, we used matrigel-coatedassays in vitro and intracranial mouse GBM model using invasive GBM8F asopposed to LN229 GBM line which is a noninvasive line (FIG. 14). oHSVand oHSV-TRAIL treatment reduced the number of GBM8F cells that hadinvaded in matrigel-coated invasion assay, with oHSV-TRAIL having astronger inhibitory effect than oHSV (FIG. 6A). In vivo, both oHSV andoHSV-TRAIL injection into intracranial GBM8F tumors reduced the numberof invading GBM8F cells as compared to the control, with oHSV-TRAILhaving a stronger inhibitory effect than oHSV (FIG. 6B). These resultsdemonstrate that oHSV-TRAIL induces apoptosis in TRAIL and oHSVresistant intracranial GBM, inhibits GBM growth and invasiveness andsignificantly increases survival in mice.

Discussion

In this study, we identified GBM lines that are either resistant toTRAIL mediated apoptosis or resistant to both oHSV-mediated oncolysisand TRAIL, and created a novel oHSV-bearing secretable-TRAIL(oHSV-TRAIL) to study the mechanism-based targeted therapy in resistantGBM lines in vitro and in vivo. We show that oHSV-TRAIL inducesapoptosis in TRAIL and oHSV resistant GBMs by targeting both cellproliferation and death pathways. Furthermore, oHSV-TRAIL inhibits GBMgrowth and invasion and prolongs survival of mice-bearing intracranialbrain tumors.

GBMs are molecularly heterogeneous tumors usually containing a subset oftumor cells that are resistant to a number of currently used anti-GBMtherapies.¹ These cells rapidly take over and ultimately result in tumorprogression. oHSV as a single agent has been tested in preclinicalstudies and shown to result in oncolysis in most GBM cells including GBMstem cells.^(13,28,29) However, while phase I clinical trials usingintracranial oHSV inoculation demonstrated safety in GBM patients, theyshowed only partial radiologic responses.^(9,30,31) These resultssuggest that GBMs might be heterogeneous in the responses to oHSV andcontain a subset of cells resistant to oHSV-mediated oncolysis. In thisstudy, we screened a panel of eight GBM cell lines that coversestablished GBM cell lines and patient derived primary GSCs. We foundthat GBM cells have differential sensitivities to oHSV-mediatedoncolysis as well as TRAIL-mediated apoptosis. To our knowledge, this isthe first study which identifies GBM lines resistant to oHSV.Interestingly, our screening indicated that the yields of oHSV do notalways parallel the efficiency of oHSV-mediated cell killing,implicating a presence of cell death mode other than oncolysis thataffects oHSV efficacy.

A number of previous studies by others and our laboratory have shownthat dual bioluminescence imaging of tumor cells and therapeuticsenables rapid and noninvasive measurement of both tumor load and fate oftherapeutics in vitro and in mice-bearing intracranialtumors.^(10,12,32,33,34) Utilizing oHSV-Fluc, GBM cells expressing Rlucand dual bioluminescence imaging, our studies reveal that two distinctbiological events, oHSV replication and its effects on cell viabilitycan be monitored. Our results indicate that oHSV can replicate in a GBMline which is resistant to oHSV, even though the viral replication andspread is greatly decreased and slow when compared to the GBM lineswhich are sensitive to oHSV. The ability of oHSV to replicate in GBMlines resistant to oHSV provides a great rationale of using oHSV todeliver cytotoxic agents like TRAIL and studying both the molecularmechanism and therapeutic effect of oHSV and TRAIL treatments in GBMsthat are resistant to oHSV-mediated oncolysis and TRAIL-mediatedapoptosis.

Various strategies have been employed to overcome resistance of tumorcells to different drug regimens. The ability of TRAIL to selectivelytarget tumor cells while remaining harmless to most normal cells makesit an attractive candidate for an apoptotic therapy for highly malignantbrain tumors. However, a large percentage of primary GBM lines areresistant to TRAIL-induced apoptosis.³⁵ Our results reveal that oHSVinfection and subsequent S-TRAIL treatment results in acaspase-3/7-mediated cell killing in a line that is resistant to bothoHSV and TRAIL. Although TRAIL is a potent tumor-specific agent, itsshort biological half-life and limited delivery across the blood-brainbarrier limit its applicability in treating brain tumors when deliveredsystemically. oHSV can replicate in situ, spread and exhibit oncolyticactivity via a direct cytocidal effect, and at the same time can deliversubstantial quantities of therapeutic molecules in situ. As compared tothe other oncolytic viruses, the capacity to incorporate largetransgenes into its genome is one of the advantages of HSV-1.³⁶ Theintratumoral delivery of oHSV-TRAIL can overcome the limitations posedby systemic administration of TRAIL as it remedies the short half-lifeof drugs, the use of multiple drug regimens to target resistant GBMs, inaddition to circumventing the restrictions posed by the blood-brainbarrier to deliver drugs to brain.

HSV like other viruses have genes such as Us3 and Rs1 (ICP4) whoseproducts have an anti-apoptotic function.^(37,38) These genes appear tofacilitate HSV replication by preventing premature cell death.Therefore, it is possible that combining proapoptotic molecule with oHSVdampens viral replication and reduces oHSV potency. Our data, however,showed that replication of oHSV and oHSV-TRAIL in glioma cells iscomparable (FIG. 10A), indicating that expression of TRAIL does notsuppress viral replication. We show that oHSV-TRAIL downregulates ERKphosphorylation, upregulates p38 and JNK phosphorylation and inducescleavage of caspases in two GBM cell lines either TRAIL or TRAIL andoHSV resistant. Downregulation of ERK phosphorylation and upregulationof P38 and JNK phosphorylation have been previously reported followinginfection with HSV-1.^(39,40) ERK1/2, members of the MAPK super family,can mediate cell proliferation and apoptosis⁴¹ while activation of ERKis primarily associated with promoting cellular proliferation.Activation of cell proliferation signals that block apoptosis isassociated with tumorigenesis and resistance to chemotherapeuticdrugs.⁴² Activation of ERK is reported to prevent Fas-induced apoptosisin activated T cells⁴³ and inhibit activation of caspases despite therelease of cytochrome c from mitochondria.^(44,45,46) In contrast toERK, the JNK and p38-MAPK signaling is activated by proinflammatorycytokines and a variety of cellular stresses, and is typically linked todifferentiation and apoptosis. Xia et al. reported that activation ofJNK and p38 and concurrent inhibition of ERK are critical for inductionof apoptosis.⁴⁷ We show that: (i) the inhibition of JNK activationsuppresses oHSV-TRAIL-mediated activation of caspase pathway, and (ii) asmall-molecule inhibitor of MEK/ERK sensitizes resistant cells toTRAIL-mediated apoptosis. Therefore, our results suggest thatoHSV-mediated downregulation of the ERK signaling and upregulation ofthe JNK and p38 signaling play a vital role in priming resistant cellsso that S-TRAIL expressed in oHSV-TRAIL infected GBM cells promotesactivation of caspase-3, -8, and -9, leading to apoptotic cell death(FIG. 7). Our studies thus underscore targeting both cell proliferationand death pathways as a crucial mechanism underlying oHSV-TRAIL-mediatedrobust induction of apoptosis in resistant GBMs.

Our in vivo studies reveal that oHSV-TRAIL results in marked attenuationof intracranial tumor growth and survival prolongation in mice-bearingTRAIL and oHSV resistant GBM. This makes a clear contrast to the lack oflong-lasting antitumor efficacy mediated by an oncolyticadenovirus-expressing TRAIL.⁴⁸ Although established GBM lines are themost commonly used models in vitro and in vivo, they fail torecapitulate the clinical properties of tumors. Given this limitation ofGBM lines as a representative GBM model, recent studies have focused onprimary GBM lines and indicated a role for tumor-initiating cells inthese lines. In an effort to test the effect of oHSV-TRAIL in suchmodels, we used GBM8F primary GBM line that contains a subpopulation ofCD133⁺ cells¹³ and exhibits highly invasive behavior in vivo. Our invitro studies showed that despite resistance to TRAIL, oHSV-TRAIL issignificantly more effective in killing and inhibiting invasiveness ofGBM8F cells than oHSV. Furthermore, the number of invading cells in vivowas strongly reduced by a single inoculation of oHSV-TRAIL intoGBM8F-generated tumors. This suggests that the oHSV spread in migratingtumor cells combined with in situ release of S-TRAIL can cooperate toblock invasiveness of these cells. Future studies will need to addresswhether this is solely due to oHSV-TRAIL-mediated increased cell deathor its additional but unknown functions that inhibit cellular migratoryor invasive machineries.

In conclusion, our findings shed a new light on targeting oHSV and TRAILresistant GBMs and pave the way for how oHSV and TRAIL can function inconcert to target both cell proliferation and death pathways inheterogeneous GBM cells. A recent promising report on oncolytic virustargeting of metastatic cancer cells of multiple cancer types in humanshighlighted the feasibility of achieving high concentrations ofanticancer molecules in situ in the context of oncolytic virus therapyTherefore, this study may provide the key to ultimately develop noveloHSV-based therapies for patients with different tumors presentingdifferent molecular profiles.

Materials and Methods

Parental and Engineered Cell Lines.

Established human GBM lines (Gli36, U87, U251, and LN229) and GBM8F weregrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum and penicillin/streptomycin. GBM stem cells (GBM4, GBM6,and BT74) were cultured in Neurobasal medium (Invitrogen, Carlsbad,Calif.) supplemented with 3 mmol/1 L-glutamine (Mediatech, Manassas,Va.), B27 (Invitrogen, Carlsbad, Calif.), 2 μg/ml heparin(Sigma-Aldrich, St Louis, Minn.), 20 ng/ml human EGF (R&D Systems,Minneapolis, Minn.), and 20 ng/ml human FGF-2 (Peprotech, Rocky Hills,N.J.) as described previously.¹³ Two lentiviral vectors were used: (i)Pico2-Fluc-mCherry, a kind gift from Dr Andrew Kung (Dana Farber CancerInstitute; Boston, Mass.), (ii) Pico2-Rluc-mCherry, which is created byligating Rluc fragment (the cDNA sequences encoding Rluc were amplifiedby PCR) into BamH1/BstB1-digested Pico2-Fluc-mcherry. Lentiviralpackaging was performed by transfection of 293T cells as previouslydescribed.³³ LN229 and GBM8F were transduced with LV-Pico2-Fluc-mCherryand LV-Pico2-Rluc-mCherry at a MOI of 2 in medium-containing protaminesulfate (2 μg/ml) and LN229-Fluc-mCherry (LN229-FmC); GBM8-Fluc-mCherry(GBM8-FmC) LN229-Rluc-mCherry (LN229-RmC); GBM8-Rluc-mCherry (GBM8-FmC)lines were obtained after puromycin (1 ug/ml) selection in culture.

Recombinant oHSVs and Viral Growth Assay.

G47ΔBAC contains the genome of G47Δ (γ34.5⁻, ICP6⁻, ICP47⁻) and acytomegalovirus promoter driven enhanced green fluorescent protein(EGFP) in place of lacZ in G47Δ.¹⁷ Recombinant oHSV vectors, G47Δ-empty(oHSV; referred to oHSV in this study), G47Δ-TRAIL (oHSV-TRAIL), andG47Δ-Fluc (oHSV-Fluc), were generated using the methods describedpreviously.^(25,26,27,50) Briefly, the respective shuttle plasmids wereintegrated into G47ΔBAC using Cre-mediated recombination in DH10BEscherichia coli, and proper recombination confirmed by restrictionanalysis of BAC clones. Next, the resulting BAC and an Flpe-expressingplasmid were cotransfected to Vero cells, to remove the BAC-derivedsequences and the EGFP gene, and allow virus to be produced. Eachrecombinant virus was plaque purified and expanded. All the recombinantoHSVs, oHSV, oHSV-TRAIL, and oHSV-Fluc, express E. coli lacZ driven byendogenous ICP6 promoter (FIG. 8A). oHSV bears no additional transgenesequences, and oHSV-TRAIL carries S-TRAIL driven by herpes simplex virusimmediate early 4/5 promoter. Cytomegalovirus immediate early promoterwas used to express Fluc-by oHSV-Fluc. For the viral growth assay, cellsplated on 96-well plates were infected with oHSV at MOI=1. After virusadsorption, media was replaced and culture continued. Forty eight hoursafter infection, culture supernatant was harvested. Titers of infectiousvirus were determined by plaque assay on Vero cells (American TypeCulture Collection, Manassas, Va.). The concentrations of TRAIL inconditioned media of GBM cells infected with oHSV-TRAIL at various MOIswere determined by ELISA using a TRAIL Immunoassay Kit (BiosourceInternational, Camarillo, Calif.) using recombinant hTRAIL expressed inE. coli as a standard.

In Vitro Bioluminescence Assays.

To determine the effects of S-TRAIL, oHSV, and oHSV-TRAIL on GBMviability and caspase activation, GBM cells were seeded on 96-wellplates (0.5×10⁴/well) and treated with different doses S-TRAIL (0-1,000ng/ml) or different MOIs of oHSV or oHSV-TRAIL 24 hours after plating.Cell viability was measured by determining the aggregate cell metabolicactivity using an ATP-dependent luminescent reagent (CellTiter-Glo;Promega, Madison, Wis.) and caspase activity was determined using aDEVD-aminoluciferin (Caspase-Glo-3/7, Promega) according tomanufacturer's instructions. For dual-luciferase imaging of GBM cellviability and oHSV-Fluc distribution in the cells, Fluc and Rlucactivity were measured in LN229-RmC and GBM8F-RmC cells by Dual-Gloluciferase assay system (Promega) according to manufacturer'sinstructions. All experiments were performed in triplicates.

Detection of Apoptosis by Flow Cytometry Using Annexin V Staining.

After a 24 hours treatment with oHSV, oHSV-TRAIL, or PBS (control),cells were stained with FITC-conjugated annexin V (Invitrogen) andpropidium iodide (2 μg/ml) in accordance with the manufacturer'sinstructions. Cells were subjected to FACS analysis with a FACSCaliber(Becton Dickinson, Franklin Lakes, N.J.). Data acquisition and analysiswere performed by CellQuest program (Becton Dickinson).

Western Blot Analysis.

Following treatment, GBM cells were lysed with NP40 buffer supplementedwith protease (Roche, Indianapolis, Ind.) and phosphatase inhibitors(Sigma-Aldrich). Twenty micrograms of harvested proteins from eachlysate were resolved on 10% SDS-PAGE, and immunoblotted with antibodiesagainst caspase-8 (Cell Signaling), cleaved PARP (Cell Signaling,Danvers, Mass.), p-44/42MAPK (ERK 1/2) (Cell Signaling),phospho-p44/42MAPK (ERK 1/2) (Thr202,Thr204) (Cell Signaling), SAPK/JNK(Cell Signaling), phospho-SAPK/JNK (Thr183/Thr185) (Cell Signaling),p38-MAPK (Cell Signaling), phospho-p38 MAPK (Cell Signaling), caspase-9(Stressgen, Pharmingdale, N.Y.), Bcl2 (Abcam, Cambridge, Mass.) orα-tubulin (Sigma-Aldrich); and blots were developed by chemiluminescenceafter incubation with horseradish peroxidase-conjugated secondaryantibodies (Santa Cruz, Santa Cruz, Calif.). Blots were then exposed tofilm (3 seconds to 5 minutes) and quantification of western blot signalswas performed using Image J. The data was normalized to α-tubulinexpression. For inhibition studies, pan-caspase inhibitor, Z-VAD-FMK(Promega) JNK inhibitor SP600125 (Sigma-Aldrich), and the MEK inhibitor,U0126, (Promega Corporation) were used.

Intracranial GBM Cell Implantation and In Vivo Bioluminescence Imaging.

To follow viral distribution in intracranial GBMs, LN229-RmC, andGBM8F-RmC GBM (5×10⁵ cells per mouse; n=3 each GBM line) werestereotactically implanted into the brains (right striatum, 2.5-mmlateral from bregma and 2.5-mm deep) of SCID mice (6 weeks of age;Charles River Laboratories, Wilmington, Mass.). Five days later,mice-bearing intracranial GBMs were injected with oHSV-Fluc (6 μl of2.0×10⁸ plaque-forming unit/ml) intratumorally at the same coordinate asthe tumor implantation, and viral distribution was followed by Flucbioluminescence imaging over time as described previously.³³ To followchanges in tumor volume and mice survival after treatment, LN229-FmC GBMcells (5×10⁵ per mouse; n=31) were stereotactically implanted into thebrains (right striatum, 2.5-mm lateral from bregma and 2.5-mm deep) ofSCID mice (6 weeks of age). Mice were imaged for the presence of tumorsby Fluc bioluminescence imaging and mice-bearing tumors were injectedwith 6 μl of 2.0×10⁸ plaque-forming unit/ml of oHSV (n=10), oHSV-TRAIL(n=10), or PBS (n=11) intratumorally. Two days post-treatment, mice (n=3in each group) were sacrificed for immunohistochemical analysis asdescribed below. Fourteen days post-treatment, mice were again injectedintratumorally with oHSV, oHSV-TRAIL, or PBS as described above. Micewere imaged for Fluc bioluminescence imaging and followed for survivaland sacrificed when neurological symptoms became apparent. All in vivoprocedures were approved by the Subcommittee on Research Animal Care atMGH.

Tissue Processing and Immunohistochemistry.

Mice were perfused by pumping ice-cold 4% paraformaldehyde directly intothe heart and the brains were fixed in 4% paraformaldehyde and frozensections were obtained for hematoxylin and eosin staining andimmunohistochemistry. 5-Bromo-4-choloro-3-indolyl-β-D-galactopyranoside(X-gal) staining was performed to identify lacZ-expressing infectedcells. For cleaved-caspase-3 staining, sections were incubated for 1hour in a blocking solution (0.3% bovine serum albumin, 8% goat serum,and 0.3% TRITON X-100™ nonionic detergent) at room temperature, followedby incubation at 4° C. overnight with anti-cleaved-caspase-3 (CellSignaling) diluted in blocking solution. Sections were incubated inAlexa Fluor 649 goat anti-rabbit secondary antibody (Invitrogen), andvisualized using confocal microscope (LSM Pascal; Zeiss, Oberkochen,Germany). The percentage of cleaved caspase-3 positive cells wascalculated by counting the positive cells in randomly selected field ofviews under a microscope.

In Vitro Invasion Assay.

The invasive capacity of GBM8F cells was tested using two chamber invitro invasion assays (BD BioCoat Matrigel Invasion Chambers). GBM8Fcells were seeded in the matrigel-coated upper chamber, infected withoHSV and oHSV-TRAIL at MOI=1 and 24 hours later the noninvading cellswere removed from the upper surface of the invasion membrane and thecells on the lower surface were stained with Diff-Quick staining kit(IMEB Inc, San Marco, Calif.). The average number of cells/field wasdetermined by counting the cells in 8 random fields/well in ×10 imagesof each well captured.

In Vivo Invasion Study.

GBM8F-FmC GBM cells (3×10⁵ per mouse; n=9) were stereotacticallyimplanted into the brain (right striatum, 2.5-mm lateral from bregma and2.5-mm deep) of SCID mice (6 weeks of age; Charles River Laboratories).Tumor-bearing mice were intratumorally injected with oHSV, oHSV-TRAIL,or PBS (n=3, each group) and 14 days post injection, mice were perfusedand brains were removed and sectioned for hematoxylin and eosin stainingand mCherry visualization. Brain sections on slides were visualized formCherry expression and the number of GBM8F tumor invading towardadjacent normal brain tissue was counted and compared in different micegroups.

Statistical Analysis.

Data were analyzed by Student t-test when comparing two groups. Datawere expressed as mean±SD and differences were considered significant atP<0.05. Kaplan-Meier analysis was used for mouse survival studies, andthe groups were compared using the log-rank test.

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What is claimed:
 1. A method of inhibiting glioblastoma tumorprogression in a subject comprising contacting a glioblastoma tumor withan effective amount of a pharmaceutical composition comprising arecombinant oncolytic herpes simplex virus comprising a nucleic acidsequence encoding, in expressible form, a secretable polypeptidecomprising the extracellular domain of tumor necrosis factor-relatedapoptosis-inducing ligand (s-TRAIL), wherein the recombinant oncolyticherpes simplex virus induces apoptosis in tumor cells that are resistantto apoptosis induction by TRAIL and by oncolytic herpes simplex virus.2. The method of claim 1, wherein the oHSV is selected from the groupconsisting of G207, G47Δ HSV-R3616, 1716, R3616, and R4009.
 3. Themethod of claim 1, wherein the TRAIL is regulated by the HSV immediateearly 4/5 promoter.
 4. The method of claim 1, wherein the virus containsan additional exogenous nucleic acid in expressible form.
 5. The methodof claim 1, wherein the virus contains no additional exogenous nucleicacids.
 6. The method of claim 1, wherein contacting is by a method ofadministration to the subject is by a method selected from the groupconsisting of intravenous administration, intraperitonealadministration, intramuscular administration, intracoronaryadministration, intraarterial administration, subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation, intracerebral, nasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue or tumor.
 7. A method of treating glioblastomain a subject comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising arecombinant oncolytic herpes simplex virus comprising a nucleic acidsequence-encoding, in expressible form, a secretable polypeptidecomprising the extracellular domain of tumor necrosis factor-relatedapoptosis-inducing ligand (s-TRAIL), wherein the recombinant oncolyticherpes simplex virus induces apoptosis in glioblastoma tumor cells thatare resistant to apoptosis induction by TRAIL and by oncolytic herpessimplex virus, to thereby treat the cancer.
 8. The method of claim 7,wherein administration is by a method selected from the group consistingof intravenous administration, intraperitoneal administration,intramuscular administration, intracoronary administration,intraarterial administration, subcutaneous administration, transdermaldelivery, intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation, intracerebral, nasal, oral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue or tumor.9. The method of claim 7, wherein the oncolytic virus is an oncolyticherpes simplex virus (oHSV) selected from the group consisting of G207,G47Δ HSV-R3616, 1716, R3616, and R4009.